WO2015068847A1 - Antigen-binding molecule containing modified antibody variable region - Google Patents
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- WO2015068847A1 WO2015068847A1 PCT/JP2014/079785 JP2014079785W WO2015068847A1 WO 2015068847 A1 WO2015068847 A1 WO 2015068847A1 JP 2014079785 W JP2014079785 W JP 2014079785W WO 2015068847 A1 WO2015068847 A1 WO 2015068847A1
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- C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
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- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Definitions
- the present invention relates to a variable region of an antibody that can bind to two different antigens (first antigen and second antigen) but does not bind to both antigens at the same time, and a third antigen different from these antigens.
- An antigen-binding molecule comprising an antibody variable region that binds to a pharmaceutical composition, a pharmaceutical composition comprising the antigen-binding molecule, and methods for producing them.
- Antibodies are attracting attention as pharmaceuticals because of their high stability in plasma and fewer side effects (Nat. Biotechnol. (2005) 23, 1073-1078 (Non-Patent Document 1) and Eur J Pharm Biopharm. (2005) 59 (3), 389-396 (Non-Patent Document 2)).
- Antibodies are not only antigen-binding, agonistic and antagonistic, but also ADCC (Antibody Dependent Cytotoxicity), ADCP (Antibody Dependent Cell phagocytosis), CDC (complementary) It induces cytotoxic activity (also referred to as effector function) by effector cells such as body-dependent cytotoxic activity).
- cytotoxic activity also referred to as effector function
- IgG1 subclass antibodies exhibit effector functions on cancer cells, many antibody drugs have been developed in the oncology region.
- Fc ⁇ RIa, Fc ⁇ RIIa, and Fc ⁇ RIIIa have a domain called ITAM (Immunoreceptor Tyrosine-based Activation Motif) in the intracellular domain and transmit an activation signal.
- ITAM Immunoreceptor Tyrosine-based Activation Motif
- Fc ⁇ RIIb has a domain called ITIM (Immunoreceptor Tyrosine-based Inhibitory Motif) in the intracellular domain and transmits an inhibitory signal.
- ITIM Immunoreceptor Tyrosine-based Inhibitory Motif
- Natural immunoglobulins bind to antigens in the variable region, and to receptors and complements such as Fc ⁇ R, FcRn, Fc ⁇ R, and Fc ⁇ R in the constant region.
- FcRn one of the binding molecules that interact in the Fc region of IgG, binds one molecule to each antibody heavy chain, and it has been reported that two molecules of FcRn bind to one IgG antibody molecule. ing.
- Fc ⁇ R interacts with the hinge region and CH2 domain of an antibody, and binds only to one molecule of an IgG type antibody (J. Bio. Chem., (20001) 276, 16469). -16477).
- This variant has a binding activity ratio (A / I ratio) to Fc ⁇ RIIIa and Fc ⁇ IIb of about 9 times that of the wild type.
- Shinkawa et al. Succeeded in increasing the binding activity to Fc ⁇ RIIIa to about 100 times by deleting the sugar chain fucose added to 297th Asn of EU numbering (J. Biol. Chem. ( 2003) 278, 3466-3473 (Non-Patent Document 10)).
- ADCC activity of human IgG1 can be greatly improved compared to natural human IgG1.
- a normal natural IgG type antibody recognizes and binds to one epitope by its variable region (Fab), and therefore can bind to only one antigen.
- Fab variable region
- many types of proteins are involved in cancer and inflammation, and the proteins may have crosstalk.
- TNF, IL1 and IL6 are involved in immune diseases (Nat. Biotech., (2011) 28, 502-10 (Non-patent Document 11)).
- other receptors are activated as one mechanism for acquiring drug resistance in cancer (EndocrocRelat Cancer (2006) 200613, 45-51 (Non-patent Document 12)). In such a case, a normal antibody that recognizes one epitope cannot inhibit a plurality of proteins.
- bispecific antibody As a molecule that inhibits multiple targets, an antibody that binds to two or more types of antigens per molecule (referred to as a bispecific antibody) has been studied. It is possible to confer binding activity to two different antigens (first antigen and second antigen) by improving the natural IgG antibody (MAbs. (2012) Mar 1, 4 (2)). Therefore, it not only has the effect of neutralizing two or more antigens with a single molecule, but also has the effect of enhancing antitumor activity by cross-linking cells with cytotoxic activity with cancer cells.
- Bispecific antibodies includes molecules that have added an antigen binding site to the N-terminus or C-terminus of the antibody (DVD-Ig or scFv-IgG), and molecules that have two different Fab regions (common L Chain bispecific antibodies and hybrid hybridomas), molecules that recognize two antigens in one Fab region (Two-in-one IgG), and molecules that use the CH3 region loop site as a new antigen binding site (Fcab) have been reported. (Nat. Rev. (2010), 10, 301-316 (Non-Patent Document 13), Peds (2010), 23 (4), 289-297 (Non-Patent Document 14)). Since any bispecific antibody interacts with Fc ⁇ R in the Fc region, the effector function of the antibody is conserved. Therefore, any antigen recognized by the Bispecific antibody binds simultaneously with Fc ⁇ R and exhibits ADCC activity against cells expressing the antigen.
- any antigen recognized by the bispecific antibody is an antigen that is specifically expressed in cancer, it recognizes one antigen because it binds to any antigen and shows cytotoxic activity against cancer cells. Efficient anticancer effect can be expected compared to conventional antibody drugs.
- any one of the antigens recognized by the Bispecific antibody is expressed in normal tissues or cells that are expressed in immune cells, normal tissue damage and cytokine release are caused by cross-linking with Fc ⁇ R. (J.JImmunol. (1999) Aug 1, 163 (3), 1246-52 (Non-patent Document 15)). As a result, strong side effects are induced.
- Catumaxomab is known as a Bispecific antibody that recognizes a protein expressed in T cells and a protein (cancer antigen) expressed in cancer cells.
- Catumaxomab binds to the CD3 ⁇ chain expressed in cancer antigen (EpCAM) and T cells by two Fabs, respectively.
- Catumaxomab induces cytotoxic activity by T cells by simultaneously binding cancer antigen and CD3 ⁇ , and induces cytotoxic activity by antigen-presenting cells such as NK cells and macrophages by simultaneously binding cancer antigen and Fc ⁇ R. .
- EpCAM cancer antigen
- Fabs cancer antigen-presenting cells
- NK cells and macrophages by simultaneously binding cancer antigen and Fc ⁇ R.
- Non-patent Document 16 Cancer Reat Rev. (2010) Oct 36 (6), 67 458-67
- an example of an antibody that reacts against cancer cells by the administration of Catumaxomab has been reported, and acquired immunity is induced (Future Oncol. (2012) Jan 8 (1), 73-85 (Non-patent Document 17)). From these results, antibodies that have both cytotoxic activity by T cells and actions by cells such as NK cells and macrophages via Fc ⁇ R (especially called trifunctional antibodies) are expected to have strong antitumor effects and induction of acquired immunity. Has been.
- trifunctional antibody binds CD3 ⁇ and Fc ⁇ R simultaneously even in the absence of cancer antigen
- CD3 ⁇ -expressing T cells and Fc ⁇ R-expressing cells are cross-linked even in the absence of cancer cells.
- various cytokines are produced in large quantities. Due to the induction of production of various cytokines independent of cancer antigens, the administration of trifunctional antibodies is currently limited to the abdominal cavity (Cancer Treat Rev. 2010 Oct 36 (6), 458-67 (non-patent literature) 16)), systemic administration is extremely difficult due to serious cytokine storm-like side effects (Cancer Immunol Immunother. 2007 Sep; 56 (9): 1397-406 (Non-patent Document 18)).
- both the antigen of cancer antigen (EpCAM) as the first antigen and the antigen of CD3 ⁇ as the second antigen can bind simultaneously with Fc ⁇ R. It is molecularly impossible to avoid such side effects due to simultaneous binding of antigen CD3 ⁇ .
- the present invention has been made in view of such circumstances, and the problem is that one variable region has binding activity to two different antigens (first antigen and second antigen).
- An antigen-binding molecule comprising a variable region of an antibody that does not bind to these antigens simultaneously and a variable region that binds to an antigen different from these antigens (third antigen), a pharmaceutical composition comprising the antigen-binding molecule,
- the present invention also provides a method for producing the antigen-binding molecule.
- variable regions of an antibody in which one variable region has binding activity to two different antigens first antigen and second antigen
- An antigen-binding molecule comprising a region and a variable region that binds to an antigen different from these antigens (third antigen), and utilizing the binding activity of the antigen-binding molecule to three different antigens
- multispecific antigen-binding molecules so far as pharmaceuticals, it avoids cross-linking between the different cells caused by binding to antigens expressed on different cells, which may cause side effects. Succeeded in producing an antigen-binding molecule capable of
- the present invention relates to the following.
- a variable region of an antibody that can bind to a first antigen and a second antigen different from the first antigen, but does not bind to the first antigen and the second antigen simultaneously, and
- An antigen binding molecule comprising a variable region that binds to a third antigen different from the first antigen and the second antigen.
- the heavy chain variable region can be bound to the first antigen and a second antigen different from the first antigen, but not simultaneously bound to the first antigen and the second antigen.
- An antigen-binding molecule comprising a variable region of an antibody in which amino acids have been modified.
- a variable region that does not bind to the first antigen and the second antigen simultaneously is a variable region that does not bind to the first antigen and the second antigen that are expressed on different cells, respectively.
- variable region of the antibody capable of binding to the first antigen and the second antigen is a variable region into which at least one amino acid modification has been introduced.
- Substitution of the amino acid sequence of the variable region that binds to the first antigen to the amino acid sequence that binds to the second antigen, or the amino acid of the variable region that binds to the first antigen The antigen-binding molecule according to [7] or [8], which is an insertion of an amino acid sequence that binds to the second antigen into the sequence.
- the antigen-binding molecule according to any one of [7] to [10], wherein the amino acid to be modified is an amino acid of a CDR1, CDR2, CDR3, or FR3 region of an antibody variable region.
- the antigen-binding molecule according to any one of [7] to [11], wherein the amino acid to be modified is a loop region amino acid.
- the amino acids to be modified include Kabat numbering 31 to 35, 50 to 65, 71 to 74 and 95 to 102 of the heavy chain variable region of the antibody, and Kabat numbering 24 to 34, 50 to 56 and 89 of the light chain variable region.
- the antigen-binding molecule according to any one of [7] to [11], which is at least one amino acid selected from -97.
- Either one of the first antigen and the second antigen is a molecule that is specifically expressed on the surface of T cells, and the other antigen is expressed on the surface of T cells or other immune cells.
- the antigen-binding molecule according to any one of [1] to [13], which is a molecule.
- Either one of the first antigen or the second antigen is CD3, and the other antigen is Fc ⁇ R, TLR, lectin, IgA, immune checkpoint molecule, TNF superfamily molecule, TNFR superfamily molecule or NK receptor
- the antigen-binding molecule according to [14] which is a molecule.
- a pharmaceutical composition comprising the antigen-binding molecule according to any one of [1] to [16] and a medically acceptable carrier.
- variable region of an antibody that can bind to the first antigen and the second antigen, but does not bind to the first antigen and the second antigen at the same time, and / or the third antigen.
- the first antigen and the second antigen that are contained in the antigen-binding molecule selected in step (ii) and that do not bind simultaneously to the first antigen and the second antigen are expressed on different cells.
- the production method according to [18] which is a variable region that does not bind to two antigens simultaneously.
- [27] Modifications from Kabat numbering 31-35, 50-65, 71-74 and 95-102 of the heavy chain variable region of the antibody, and Kabat numbering 24-34, 50-56 and 89-97 of the light chain variable region
- Either one of the first antigen and the second antigen is a molecule that is specifically expressed on the surface of T cells, and the other antigen is expressed on the surface of T cells or other immune cells.
- Either one of the first antigen and the second antigen is CD3, and the other antigen is Fc ⁇ R, TLR, IgA, lectin, immune checkpoint molecule, TNF superfamily molecule, TNFR superfamily molecule or NK receptor
- the production method according to [28] which is a molecule.
- the production method according to [28] or [29], wherein the third antigen is a molecule expressed specifically in cancer tissue.
- a method for treating cancer comprising a step of administering the antigen-binding molecule according to any one of [1] to [16].
- the “variable region of an antibody” usually means a region composed of four framework regions (FR) and three complementarity-determining regions (CDRs) sandwiched between them. As long as it has an activity of binding to a part or all of the partial sequence, the partial sequence is also included. Particularly preferred is a region comprising an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
- the variable region of the antibody of the present invention may be of any sequence, mouse antibody, rat antibody, rabbit antibody, goat antibody, camel antibody, humanized antibody obtained by humanizing these non-human antibodies, and human It may be a variable region of an antibody of any origin, such as an antibody.
- Humanized antibody refers to an antibody derived from a mammal other than a human, also referred to as a reshaped human antibody, such as a complementarity determination region (CDR) of a mouse antibody to the CDR of a human antibody. It is transplanted.
- CDR complementarity determination region
- Methods for identifying CDRs are known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md .; Chothia et al., Nature (1989) 342: 877) .
- general gene recombination techniques are also known (see European Patent Application Publication No. EP-125023 and WO96 / 02576).
- variable region of the antibody of the present invention does not bind to the first antigen and the second antigen at the same time in the state where the variable region of the antibody of the present invention is bound to the first antigen. This means that it cannot bind to the second antigen, and conversely, when the variable region is bound to the second antigen, it cannot bind to the first antigen.
- does not bind to the first antigen and the second antigen at the same time means that the two cells of the cell expressing the first antigen and the cell expressing the second antigen are cross-linked. Or not simultaneously binding to the first and second antigens expressed in separate cells.
- variable region of such an antibody is not particularly limited as long as it has the function.
- the variable region of a variable region of an IgG-type antibody may be modified so that it binds to a desired antigen.
- An area can be mentioned.
- the amino acid to be modified for example, an amino acid that does not lose binding to the antigen by amino acid modification is selected from the variable region of the antibody that binds to the first antigen or the second antigen.
- “expressed on different cells” is only required to be expressed on different cells.
- a T cell and another T cell can be the same type of cell. There may be different types of cells such as T cells and NK cells.
- the amino acid modification of the present invention may be used alone or in combination.
- the number of combinations is not particularly limited, and can be set as appropriate within the range in which the object of the invention can be achieved. For example, 2 to 30 or less, preferably 2 to 25 or less, 2 2 or more and 20 or less, 2 or more and 15 or less, 2 or more and 10 or less, 2 or more and 5 or less, 2 or more and 3 or less.
- the amino acid modification may be added only to the heavy chain variable region or the light chain variable region of the antibody, or may be appropriately distributed to both the heavy chain variable region and the light chain variable region.
- the binding activity of the antibody before modification is maintained, for example, 50% or more, preferably 80% or more, more preferably compared to before modification.
- the binding activity may be increased by amino acid modification.
- the binding activity may be 2 times, 5 times, 10 times, etc., compared to before the modification.
- regions for amino acid modification include a region exposed to the solvent in the variable region and a loop region.
- CDR1, CDR2, CDR3, FR3 region and loop region are preferable.
- Kabat numbering 31 to 35, 50 to 65, 71 to 74, 95 to 102 of the heavy chain variable region, Kabat numbering 24 to 34, 50 to 56, 89 to 97 of the light chain variable region are preferable.
- More preferred are Kabat numbering 31, 52a-61, 71-74, 97-101 of the chain variable region, and Kabat numbering 24-34, 51-56, 89-96 of the L chain variable region.
- an amino acid that increases the binding activity with the antigen may be introduced together.
- the “loop region” means a region where there are residues that are not involved in maintaining the ⁇ -barrel structure of immunoglobulin.
- the amino acid modification means any one of substitution, deletion, addition, insertion, modification, or a combination thereof.
- amino acid modification can be rephrased as amino acid mutation and is used interchangeably.
- the purpose is to modify, for example, the following points (a) to (c) by substituting with another amino acid residue.
- Amino acid residues are classified into the following groups based on general side chain properties: (1) Hydrophobicity: norleucine, met, ala, val, leu, ile; (2) Neutral hydrophilicity: cys, ser , Thr, asn, gln; (3) Acidity: asp, glu; (4) Basicity: his, lys, arg; (5) Residues that affect chain orientation: gly, pro; and (6) Aromatic Sex: trp, tyr, phe.
- substitution of amino acid residues within each of these groups is called conservative substitution, while substitution of amino acid residues between other groups is called non-conservative substitution.
- the substitution in the present invention may be a conservative substitution, a non-conservative substitution, or a combination of a conservative substitution and a non-conservative substitution.
- variable regions of antibodies that bind to the first antigen or the second antigen amino acids that do not lose binding to the antigen by amino acid modification are randomly modified.
- Select a variable region that can bind to the first antigen and the second antigen, but cannot bind at the same time, or have a binding activity to the desired antigen in advance are modifications that insert a known peptide into the above region. Examples of peptides known to have a binding activity for a desired antigen in advance include the peptides shown in Table 1.
- the first antigen and a second antigen different from the first antigen can be bound, but the heavy antigen is not so bound to the first antigen and the second antigen at the same time.
- An antigen-binding molecule is provided that comprises an antibody variable region in which the amino acids of the chain variable region have been modified. For example, by introducing the above-described amino acid alteration (substitution, deletion, addition, insertion, or modification, or a combination thereof) into the heavy chain variable region, the first antigen and the first antigen An antibody variable region can be created that can bind to different second antigens but does not bind to the first and second antigens simultaneously.
- the position for introducing an amino acid modification is preferably a heavy chain variable region, and more preferable regions include a region exposed to a solvent in the variable region and a loop region.
- CDR1, CDR2, CDR3, FR3 region and loop region are preferable.
- Kabat numbering 31 to 35, 50 to 65, 71 to 74, 95 to 102 of the heavy chain variable region is preferred, and Kabat numbering 31, 52a to 61, 71 to 74, 97 to 101 of the heavy chain variable region is preferable. Is more preferable.
- an amino acid that increases the binding activity with the antigen may be introduced together.
- variable region of the antibody of the present invention may be combined with known modifications in addition to the above modifications.
- modification of pyroglutamic acid by pyroglutamylation of N-terminal glutamine of the variable region is a modification well known to those skilled in the art. Therefore, the antibody of the present invention comprises a variable region in which the heavy chain is modified with pyroglutamic acid when the N-terminus of the heavy chain is glutamine.
- variable region of the antibody of the present invention may be capable of repeatedly binding to the antigen by adding a modification having pH-dependent binding properties to the antigen (WO / 2009/125825).
- an amino acid modification that changes the binding activity to the antigen according to the concentration of the target tissue-specific compound can be added to the variable region that binds to the third antigen of these antibodies (WO2013 / 180200). ).
- modification of the variable region can increase binding activity, improve specificity, decrease pI, impart pH-dependent properties to antigen binding, improve binding thermal stability, improve solubility, modify chemical modification Stability, improved heterogeneity derived from sugar chains, avoidance of T cell epitopes identified using in silico prediction to reduce immunogenicity, or identified in assays using in vitro T cells, Alternatively, modifications aimed at introducing a T cell epitope that activates regulatory T cells can be performed (mAbs 3: 243-247, 2011).
- variable region of the antibody of the present invention can bind to the first antigen and the second antigen can be measured using a known method. For example, it can be measured by an electrochemiluminescence method (ECL method) (BMC Research Notes 2011, 4: 281).
- ECL method electrochemiluminescence method
- a region capable of binding to the first antigen and the second antigen of a test antigen-binding molecule labeled with biotin for example, a low molecular weight antibody consisting of a Fab region, or monovalent 1st or 2nd antigen labeled with a sulfo-tag (Ru complex) is mixed with a natural antibody (an antibody that does not have one of the two Fab regions of a normal antibody), and a streptavidin solid phase Add onto the crystallization plate. At this time, the test antigen-binding molecule labeled with biotin is bound to streptavidin on the plate.
- a natural antibody an antibody that does not have one of the two Fab regions of a normal antibody
- the first antigen or the second antigen and the above-mentioned region of the test antigen binding molecule can be confirmed. It can also be measured by ELISA, FACS (fluorescence activated cell sorting), ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), BIACORE method using surface plasmon resonance (SPR) phenomenon, etc. (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).
- Biacore® (GE® Healthcare), which is an interaction analysis device using the surface plasmon resonance (SPR) phenomenon.
- Biacore includes all models such as Biacore T100, T200, X100, A100, 4000, 3000, 2000, 1000, and C.
- Any sensor chip for Biacore such as CM7, CM5, CM4, CM3, C1, SA, NTA, L1, HPA, and Au chip can be used as the sensor chip.
- Proteins for supplementation such as chain antibodies, anti-human Fc antibodies, antigen proteins, and antigen peptides are immobilized.
- the first antigen or the second antigen is flowed there as an analyte, the interaction is measured, and a sensorgram is obtained.
- the concentration of the first antigen or the second antigen at this time can be carried out in the range of several ⁇ M to several pM according to the strength of interaction such as KD of the sample to be measured.
- the antigen-binding molecule not the antigen-binding molecule but the first antigen or the second antigen can be immobilized on the sensor chip, and the antibody sample to be evaluated can be allowed to interact therewith.
- KD dissociation constant
- ALPHA screen is implemented based on the following principle by ALPHA technology using two beads of donor and acceptor.
- a luminescent signal is detected only when the molecule bound to the donor bead interacts biologically with the molecule bound to the acceptor bead and the two beads are in close proximity.
- a photosensitizer in the donor bead excited by the laser converts ambient oxygen into excited singlet oxygen. Singlet oxygen diffuses around the donor bead, and when it reaches the adjacent acceptor bead, it causes a chemiluminescence reaction in the bead, and finally light is emitted.
- the chemiluminescence reaction does not occur because the singlet oxygen produced by the donor bead does not reach the acceptor bead.
- the Biacore system takes the shift amount, that is, the mass change at the sensor chip surface on the vertical axis, and displays the time change of mass as measurement data (sensorgram).
- the amount of analyte binding to the ligand captured on the sensor chip surface from the sensorgram (the amount of change in the response on the sensorgram before and after the interaction of the analyte) is determined.
- the amount of binding also depends on the amount of ligand, it is necessary to compare under the condition that the amount of ligand can be regarded as essentially the same amount.
- the kinetics: association rate constant (ka) and dissociation rate constant (kd) are obtained from the curve of the sensorgram, and the affinity (KD) is obtained from the ratio of the constants.
- an inhibition measurement method is also preferably used. Examples of inhibition assays are described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.
- the antigen-binding molecule of the present invention "does not bind to the first antigen and the second antigen at the same time" is confirmed after having confirmed the binding activity to the first antigen and the second antigen. Whether the antigen-binding molecule containing the variable region having the binding activity has binding activity for the remaining one after binding either the first antigen or the second antigen in advance. Can be confirmed by measuring using the method described above. In addition, measuring whether the binding of the antigen-binding molecule to either the first antigen or the second antigen immobilized on the ELISA plate or the sensor chip is inhibited by adding the other to the solution. Can also be confirmed.
- a first antigen labeled with a sulfo-tag (Ru complex) and a second antigen unlabeled are prepared in a test antigen-binding molecule labeled with biotin.
- the test antigen-binding molecule can bind to the first antigen and the second antigen, but does not bind simultaneously with the first antigen and the second antigen, in the absence of the unlabeled second antigen.
- the luminescence signal is detected.
- the luminescence signal decreases. Relative binding activity can be determined by quantifying this decrease in signal issued.
- the test antigen-binding molecule interacts with the first antigen to generate a signal of 520-620 nm.
- the untagged second antigen competes with the interaction between the test antigen binding molecule and the first antigen.
- Relative binding activity can be determined by quantifying the decrease in fluorescence that results from competition. It is known that a polypeptide is biotinylated using Sulfo-NHS-biotin or the like.
- the first antigen is expressed in a cell or the like holding a vector capable of expressing a fusion gene in which a polynucleotide encoding the first antigen and a polynucleotide encoding GST are fused in frame.
- a purification method using a glutathione column can be appropriately employed.
- the obtained signal is suitably analyzed by fitting to a one-site competition model using nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego). At this time, the same analysis can be performed by tagging the second antigen and not tagging the first antigen. Further, a method using fluorescence resonance energy transfer (FRET) can be used.
- FRET fluorescence resonance energy transfer
- FRET is a phenomenon in which excitation energy moves directly between two adjacent fluorescent molecules by electron resonance.
- the excitation energy of the donor (excited fluorescent molecule) is transferred to the acceptor (another fluorescent molecule in the vicinity of the donor), and the fluorescence emitted from the donor disappears (exactly the fluorescence lifetime). Instead, fluorescence is emitted from the acceptor instead. It is possible to analyze whether this is a dual-Fab using this phenomenon. For example, when the first antigen introduced with a fluorescent donor and the second antigen introduced with a fluorescent acceptor are simultaneously bound to a test antigen-binding molecule, the donor's fluorescence disappears and the acceptor emits fluorescence. A change in fluorescence wavelength occurs.
- Such an antibody is judged not to be dual-Fab.
- the test antigen binding molecule when the first antigen, the second antigen, and the test antigen binding molecule are mixed, if the fluorescence wavelength of the fluorescent donor bound to the first antigen does not change, the test antigen binding molecule is It can be said that it is dual-Fab.
- a test antigen-binding molecule labeled with biotin on the donor bead is bound to streptavidin on the donor bead, and a first antigen tagged with glutathione S-transferase (GST) is bound to the acceptor bead.
- GST glutathione S-transferase
- the untagged second antigen competes with the interaction between the test antigen binding molecule and the first antigen.
- Relative binding activity can be determined by quantifying the decrease in fluorescence that results from competition.
- a polypeptide is biotinylated using Sulfo-NHS-biotin or the like.
- the first antigen is expressed in a cell or the like holding a vector capable of expressing a fusion gene in which a polynucleotide encoding the first antigen and a polynucleotide encoding GST are fused in frame.
- a purification method using a glutathione column can be appropriately employed.
- the obtained signal is suitably analyzed by fitting to a one-site competition model using nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).
- the tagging is not limited to GST, and any tag such as histidine tag, MBP, CBP, Flag tag, HA tag, V5 tag, c-myc tag, etc. may be used.
- the binding of the test antigen binding molecule to the donor bead is not limited to the binding using the biotin-streptavidin reaction.
- the test antigen-binding molecule contains Fc
- a method of binding the test antigen-binding molecule via an Fc recognition protein such as Protein A or Protein G on the donor bead can be considered.
- the first antigen and the second antigen when they are not expressed on the cell membrane like a soluble protein, or when both are present on the same cell, the first antigen and the second antigen Can be simultaneously bound to both antigens, but when they are expressed on different cells, the case where they cannot be bound simultaneously can also be measured by using a known method. Specifically, cells that express the first antigen and cells that express the second antigen, respectively, even when positive by ECL-ELISA that detects simultaneous binding to the first antigen and the second antigen. When the test antigen-binding molecules are mixed, if these three do not bind simultaneously, it can be shown that they cannot bind simultaneously when expressed on different cells. For example, it can be measured by an ECL-ELISA method using cells.
- Cells that express the first antigen are immobilized on a plate in advance and bound to the test antigen-binding molecule, and then cells that express the second antigen are added.
- a sulfo-tag-labeled antibody to detect another antigen that is expressed only in cells that express the second antigen, the signal is displayed when the two antigens expressed on the two cells are bound simultaneously. If observed and not bound simultaneously, no signal is observed. Alternatively, it can be measured by the alphascreen method.
- test antigen-binding molecule When a test antigen-binding molecule is mixed with a cell expressing a first antigen bound to a donor bead, a cell expressing a second antigen bound to an acceptor bead, A signal is observed when bound simultaneously with two antigens, and no signal is observed when bound simultaneously. Alternatively, it can be measured by an interaction analysis method using Octet. First, cells expressing a first antigen to which a peptide tag has been added are bound to a biosensor that recognizes a peptide tag.
- binding activity measurement based on biological activity is possible instead of binding activity.
- a cell that expresses a first antigen, a cell that expresses a second antigen, and a test antigen-binding molecule are mixed and cultured, the two antigens expressed on two cells are bound simultaneously. Since they are mutually activated via the test antigen-binding molecule, changes in the activation signal such as an increase in phosphorylation downstream of each antigen can be detected.
- cytokine production is induced as a result of activation, it is possible to determine whether or not to bind to two cells simultaneously by measuring the amount of cytokine production.
- the “Fc region” refers to a region containing a fragment consisting of a hinge region or a part thereof, CH2 and CH3 domains in an antibody molecule.
- the Fc region of the IgG class is EU numbering (also referred to herein as EU INDEX) and means, for example, from the 226th cysteine to the C terminus, or from the 230th proline to the C terminus, but is not limited thereto.
- Fc region is suitably obtained by re-elution of the fraction adsorbed on the protein A column or protein G column after partial digestion of IgG1, IgG2, IgG3, IgG4 monoclonal antibody, etc. with a protease such as pepsin Can be done.
- proteolytic enzymes are not particularly limited as long as full-length antibodies can be digested so that Fab and F (ab ') 2 can be produced in a limited manner by appropriately setting the reaction conditions of the enzyme such as pH.
- pepsin, papain, etc. can be illustrated.
- the “antigen-binding molecule” in the present invention is not particularly limited as long as it contains the “antibody variable region” of the present invention, and further includes peptides and proteins having a length of about 5 amino acids or more. Also good.
- the polypeptide is not limited to biologically derived peptides or proteins, and may be, for example, a polypeptide having an artificially designed sequence. Moreover, any of natural polypeptide, synthetic polypeptide, recombinant polypeptide, etc. may be sufficient.
- an antigen-binding molecule containing an antibody Fc region can be mentioned.
- an Fc region derived from natural IgG can be used.
- the natural IgG means a polypeptide belonging to the class of antibodies that includes the same amino acid sequence as IgG found in nature and is substantially encoded by an immunoglobulin gamma gene.
- natural human IgG means natural human IgG1, natural human IgG2, natural human IgG3, natural human IgG4, and the like.
- Naturally-occurring IgG includes naturally occurring mutants.
- human IgG1 As constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, a plurality of allotype sequences due to gene polymorphisms are described in Sequences of proteins of immunological interest, NIH Publication No.91-3242. Any of them may be used.
- sequence of human IgG1 may be DEL or EEM as the amino acid sequence of EU numbering 356-358.
- Fc regions of antibodies include IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM type Fc regions.
- an Fc region derived from a natural human IgG antibody can be used.
- a constant region of natural IgG specifically, a constant region originating from natural human IgG1 (SEQ ID NO: 1), a constant region originating from natural human IgG2 (sequence) No.
- the Fc region of the present invention is particularly preferably an Fc region with reduced binding activity to the Fc ⁇ receptor.
- the Fc ⁇ receptor (which may be described as Fc ⁇ receptor, Fc ⁇ R or Fc ⁇ receptor in the present specification) refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, or IgG4. By any member of the family of proteins encoded by the Fc ⁇ receptor gene.
- this family includes Fc ⁇ RI (CD64), including isoforms Fc ⁇ RIa, Fc ⁇ RIb and Fc ⁇ RIc; isoforms Fc ⁇ RIIa (including allotypes H131 (H) and R131 (R)), Fc ⁇ RIIb (Fc ⁇ RIIb-1 and Fc ⁇ RIIb- Fc ⁇ RII (CD32) including Fc ⁇ RIIc (including 2) and Fc ⁇ RIII (CD16) including isoforms Fc ⁇ RIIIa (including allotypes V158 and F158) and Fc ⁇ RIIIb (including allotypes Fc ⁇ RIIIb-NA1 and Fc ⁇ RIIIb-NA2), and any undiscovered Human Fc ⁇ Rs or Fc ⁇ R isoforms or allotypes, but are not limited to these.
- Fc ⁇ R includes, but is not limited to, those derived from human, mouse, rat, rabbit and monkey, and may be derived from any organism.
- Mouse Fc ⁇ Rs include Fc ⁇ RI (CD64), Fc ⁇ RII (CD32), Fc ⁇ RIII (CD16) and Fc ⁇ RIII-2 (CD16-2), as well as any undiscovered mouse Fc ⁇ Rs or Fc ⁇ R isoforms or allotypes. It is not limited to. Suitable examples of such Fc ⁇ receptors include human Fc ⁇ RI (CD64), Fc ⁇ RIIa (CD32), Fc ⁇ RIIb (CD32), Fc ⁇ RIIIa (CD16) and / or Fc ⁇ RIIIb (CD16).
- Fc ⁇ R has an active receptor having an ITAM (immunoreceptor tyrosine-based activation motif) and an inhibitory receptor having an ITIM (immunoreceptor tyrosine-based inhibitory motif).
- Fc ⁇ R is classified into Fc ⁇ RI, Fc ⁇ RIIa R, Fc ⁇ RIIa H, Fc ⁇ RIIIa, and Fc ⁇ RIIIb active Fc ⁇ R, and Fc ⁇ RIIb inhibitory Fc ⁇ R.
- the polynucleotide sequence and amino acid sequence of Fc ⁇ RI are NM_000566.3 and NP_000557.1, respectively.
- the polynucleotide sequence and amino acid sequence of Fc ⁇ RIIa are BC020823.1 and AAH20823.1, respectively.
- the polynucleotide sequence and amino acid sequence of Fc ⁇ RIIb are BC146678.1 and AAI46679.1, respectively,
- Fc ⁇ RIIIa polynucleotide sequence and amino acid sequence are BC033678.1 and AAH33678.1, respectively
- Fc ⁇ RIIIb polynucleotide sequence and amino acid sequence are BC128562.1 and AAI28563.1, respectively. It is listed (RefSeq registration number).
- Fc ⁇ RIIa has two gene polymorphisms in which the 131st amino acid of Fc ⁇ RIIa is substituted with histidine (H type) or arginine (R type) (J. Exp. Med, 172, 19-25, 1990).
- Fc ⁇ RIIb has two gene polymorphisms in which the 232nd amino acid of Fc ⁇ RIIb is replaced with isoleucine (type I) or threonine (type T) (Arthritis. Rheum. 46: 1242-1254 (2002) ).
- Fc ⁇ RIIIa has two gene polymorphisms in which the 158th amino acid of Fc ⁇ RIIIa is substituted with valine (V type) or phenylalanine (F type) (J. Clin. Invest. 100 (5): 1059 -1070 (1997)).
- Fc ⁇ RIIIb has two gene polymorphisms, NA1 type and NA2 type (J. Clin. Invest. 85: 1287-1295 (1990)).
- Fc ⁇ receptor binding activity is reduced should be confirmed by well-known methods such as FACS, ELISA format, ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) and BIACORE method using surface plasmon resonance (SPR) phenomenon.
- FACS Fluorescence Activated Cell Sorting
- ELISA format ALPHA screen
- SPR surface plasmon resonance
- the ALPHA screen is implemented according to the following principle by ALPHA technology using two beads of donor and acceptor. A molecule bound to the donor bead interacts biologically with the molecule bound to the acceptor bead, and a luminescent signal is detected only when the two beads are in close proximity.
- a photosensitizer in the donor bead excited by the laser converts ambient oxygen into excited singlet oxygen.
- Singlet oxygen diffuses around the donor bead, and when it reaches the adjacent acceptor bead, it causes a chemiluminescence reaction in the bead, and finally light is emitted.
- the chemiluminescence reaction does not occur because the singlet oxygen produced by the donor bead does not reach the acceptor bead.
- an antigen-binding molecule labeled with biotin is bound to the donor bead, and an Fc ⁇ receptor tagged with glutathione S-transferase (GST) is bound to the acceptor bead.
- GST glutathione S-transferase
- antigen-binding molecules with wild-type Fc regions interact with Fc ⁇ receptors to produce signals of 520-620 nm.
- An antigen-binding molecule having a mutated Fc region that is not tagged competes with the interaction between an antigen-binding molecule having a wild-type Fc region and the Fc ⁇ receptor. Relative binding affinity can be determined by quantifying the decrease in fluorescence that results from competition.
- biotinylate antigen-binding molecules such as antibodies using Sulfo-NHS-biotin or the like.
- a method of tagging Fc ⁇ receptor with GST it is expressed in a cell or the like holding a vector capable of expressing a fusion gene in which a polynucleotide encoding Fc ⁇ receptor and a polynucleotide encoding GST are fused in frame.
- a method of purification using a glutathione column can be appropriately employed.
- the obtained signal is suitably analyzed by fitting to a one-site competition model using nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).
- the Biacore system takes the shift amount, that is, the mass change at the sensor chip surface on the vertical axis, and displays the time change of mass as measurement data (sensorgram).
- Kinetics association rate constant (ka) and dissociation rate constant (kd) are obtained from the sensorgram curve, and affinity (KD) is obtained from the ratio of the constants.
- an inhibition measurement method is also preferably used. Examples of inhibition assays are described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.
- the fact that the binding activity to the Fc ⁇ receptor is reduced is, for example, based on the above analysis method, compared to the binding activity of the antigen-binding molecule containing the Fc region as a control, compared to the test antigen binding Binding activity of molecules is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, 15% or less, particularly preferably 10% or less, 9% or less, 8% or less 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.
- an antigen-binding molecule having an Fc region of IgG1, IgG2, IgG3 or IgG4 monoclonal antibody can be used as appropriate.
- the structure of the Fc region is as follows. RefSeq registration number CAA27268.1 with an A added at the N terminus), SEQ ID NO: 4 (RefSeq registration number AAB59394.1 with an N added at the N ending).
- an antigen-binding molecule having a variant of the Fc region of an antibody of a specific isotype is used as a test substance
- the antigen-binding molecule having the Fc region of the antibody of the specific isotype is used as a control.
- the effect of the mutation of the mutant on the binding activity to the Fc ⁇ receptor is verified.
- an antigen-binding molecule having a variant of the Fc region that has been verified to have reduced binding activity to the Fc ⁇ receptor is appropriately prepared.
- variants include deletion of amino acids 231A-238S identified according to EU numbering (WO 2009/011941), C226S, C229S, P238S, (C220S) (J. Rheumatol (2007) 34, 11), C226S, C229S (Hum.Antibod.Hybridomas (1990) 1 (1), 47-54), C226S, C229S, E233P, L234V, L235A (Blood (2007) 109, 1185-1192) It is known.
- any one of the following amino acids specified according to EU numbering positions 220, 226, 229, 231, 232, 233, 234 , 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 297, 298, 299
- Preferred examples include antigen-binding molecules having Fc regions in which the position, position 300, position 325, position 327, position 328, position 329, position 330, position 331, and position 332 are substituted.
- the isotype of the antibody that is the origin of the Fc region is not particularly limited, and an Fc region originating from an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be used as appropriate, but an Fc region originating from a natural human IgG1 antibody It is preferably used.
- amino acids constituting the Fc region of IgG1 antibody one of the following substitutions specified according to EU numbering (position of amino acid residue specified according to EU numbering, one-letter amino acid positioned before the number) The symbol is the amino acid residue before substitution, and the one-letter amino acid symbol located after the number represents the amino acid residue before substitution); (A) L234F, L235E, P331S, (B) C226S, C229S, P238S, (C) C226S, C229S, (D) C226S, C229S, E233P, L234V, L235A Or an antigen-binding molecule having an Fc region in which the amino acid sequence from positions 231 to 238 has been deleted can be used as appropriate.
- amino acids constituting the Fc region of IgG2 antibody one of the following substitutions specified according to EU numbering (position of amino acid residue specified according to EU numbering, one-letter amino acid positioned before the number) The symbol is the amino acid residue before substitution, and the one-letter amino acid symbol located after the number represents the amino acid residue before substitution); (E) H268Q, V309L, A330S, P331S (F) V234A (G) G237A (H) V234A, G237A (I) A235E, G237A (J) V234A, A235E, G237A Antigen-binding molecules having an Fc region that has been subjected to can be used as appropriate.
- amino acids constituting the Fc region of IgG3 antibody one of the following substitutions specified according to EU numbering (number of amino acid residue specified according to EU numbering, one-letter amino acid located before the number) The symbol is the amino acid residue before substitution, and the one-letter amino acid symbol located after the number represents the amino acid residue before substitution); (K) F241A (L) D265A (M) V264A Antigen-binding molecules having an Fc region that has been subjected to can be used as appropriate.
- amino acids constituting the Fc region of IgG4 antibody one of the following substitutions specified according to EU numbering (position of amino acid residue specified according to EU numbering, one-letter amino acid located before the number) The symbol is the amino acid residue before substitution, and the one-letter amino acid symbol located after the number represents the amino acid residue before substitution); (N) L235A, G237A, E318A (O) L235E (P) F234A, L235A Antigen-binding molecules having an Fc region that has been subjected to can be used as appropriate.
- any one of the following amino acids specified according to EU numbering 233, 234, 235, 236, 237, 327
- examples include antigen-binding molecules having Fc regions in which the EU numbering is substituted with the corresponding amino acids in the corresponding IgG2 or IgG4 at positions 330, 331.
- any one or more of the following amino acids specified according to EU numbering; positions 234, 235, and 297 are the other amino acids.
- Preferred examples include antigen-binding molecules having an Fc region substituted with an amino acid.
- the type of amino acid present after the substitution is not particularly limited, but an antigen-binding molecule having an Fc region in which any one or more amino acids at positions 234, 235, and 297 are substituted with alanine is particularly preferable.
- any one of the following amino acids specified according to EU numbering; an antigen-binding molecule having an Fc region substituted at position 265 with another amino acid Preferably mentioned.
- the type of amino acid present after the substitution is not particularly limited, but an antigen-binding molecule having an Fc region in which the amino acid at position 265 is substituted with alanine is particularly preferable.
- a multispecific antibody containing the variable region of the antibody of the present invention can be mentioned.
- undesired heavy chains are introduced by introducing a charge repulsion at the interface of the second constant region (CH2) of the antibody heavy chain or the third constant region (CH3) of the heavy chain.
- a technique for suppressing the association between each other can be applied (WO2006 / 106905).
- amino acid residues that contact at the interface of other constant regions of the H chain include, for example, EU in the CH3 region Numbering region 356, EU numbering 439th residue, EU numbering 357th residue, EU numbering 370th residue, EU numbering 399th residue, EU numbering 409th residue Can be mentioned.
- a group to three sets of amino acid residues can be an antibody having the same kind of charge;) (1) amino acid residues contained in the H chain CH3 region, and amino acid residues at positions 356 and 439 of EU numbering; (2) Amino acid residues contained in the H chain CH3 region, amino acid residues at positions 357 and 370 of the EU numbering, and (3) Amino acid residues contained in the H chain CH3 region, comprising the EU numbering at position 399. And amino acid residue at position 409.
- a set of amino acid residues selected from the set of amino acid residues shown in the above (1) to (3) in a second H chain CH3 region different from the first H chain CH3 region are the first H chain CH3 region.
- the antibody may have an opposite charge to the corresponding amino acid residue in.
- amino acid residues described in (1) to (3) above are close to each other when they are associated.
- a person skilled in the art finds a site corresponding to the amino acid residue described in (1) to (3) above by using homology modeling using commercially available software for the desired H chain CH3 region or H chain constant region.
- the amino acid residue at the site can be subjected to modification.
- the “charged amino acid residue” is preferably selected from, for example, amino acid residues included in any of the following groups (a) or (b); (A) glutamic acid (E), aspartic acid (D), (B) Lysine (K), arginine (R), histidine (H).
- “having the same kind of charge” means, for example, that two or more amino acid residues each have an amino acid residue included in any one group of (a) or (b). Means that. “Having an opposite charge” means, for example, an amino acid residue in which at least one amino acid residue of two or more amino acid residues is included in any one group of the above (a) or (b) Means that the remaining amino acid residues have amino acid residues contained in different groups.
- the first H chain CH3 region and the second H chain CH3 region may be cross-linked by a disulfide bond.
- the amino acid residues to be modified in the present invention are not limited to the above-described antibody variable region or antibody constant region amino acid residues. A person skilled in the art can find amino acid residues that form an interface for polypeptide variants or heterologous multimers by homology modeling using commercially available software, etc. Amino acid residues can be subjected to modification.
- a technique for producing a bispecific antibody using two bacterial cell lines that respectively express one chain of an antibody consisting of one H chain and one L chain can also be used.
- a light chain that forms a variable region that binds to the first epitope and a light chain that forms a variable region that binds to the second epitope are each bound to the first epitope.
- CrossMab technology (Scaefer et al. (Proc. Natl. Acad. Sci. Sci.), Known as the association of heterogeneous light chains that associate with the heavy chain that forms the variable region and the heavy chain that forms the variable region that binds to the second epitope.
- the heterogeneity of antibodies can be achieved by placing monoclonal antibodies substituted with specific amino acids in the heavy chain CH3 region under reducing conditions. And a method for obtaining a desired bispecific antibody.
- Preferable amino acid substitution sites in the method include, for example, the EU numbering 392rd residue and EU numbering 397th residue in the CH3 region.
- Dual-characteristic antibodies can also be made; (1) amino acid residues contained in the H chain CH3 region, EU numbering amino acids 356 and 439, (2) contained in the H chain CH3 region Amino acid residues, EU numbering amino acid residues at positions 357 and 370, (3) amino acid residues contained in the H chain CH3 region, EU numbering amino acid residues at positions 399 and 409.
- a set of amino acid residues selected from the set of amino acid residues shown in the above (1) to (3) in a second H chain CH3 region different from the first H chain CH3 region One to three amino acid residues corresponding to the amino acid residue groups shown in (1) to (3) having the same kind of charge in the first H chain CH3 region are the first H chain CH3 region.
- An antibody having a charge opposite to that of the corresponding amino acid residue in can be used to produce a dual-characteristic antibody.
- the multispecific antibody of the present invention can also be obtained by separating and purifying the target multispecific antibody from the produced antibody. It is possible to obtain sex antibodies. For example, by introducing amino acid substitutions in the variable regions of two types of H chains and adding isoelectric point differences, a method has been reported that makes it possible to purify two types of homozygote and the desired heteroantibody by ion exchange chromatography. (WO2007114325).
- a method for purifying heterozygotes a method for purifying a heterodimerized antibody consisting of a mouse IgG2a H chain that binds to protein A and a rat IgG2b H chain that does not bind to protein A by using protein A so far has been reported (WO98050431, WO95033844). Furthermore, by using H chains in which the amino acid residues at the 435th and 436th EU numbering, which are the binding sites of IgG and Protein A, are substituted with amino acids having different binding powers to Protein A such as Tyr and His, By changing the interaction with Protein A and using a Protein A column, it is possible to efficiently purify only the heterodimerized antibody.
- the antigen-binding molecule of the present invention may be prepared by separately producing an antigen-binding molecule having the same amino acid sequence based on the above-described modification.
- the amino acid sequence can be modified by various methods known in the art. These methods include, but are not limited to, site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide- directed dual amber method for site-directed mutagenesis. Gene 152, 271-275, Zoller, MJ, and Smith, M.
- the “antigen-binding molecule” of the present invention includes both the heavy and light chains that form the “antibody variable region” of the present invention within a single polypeptide chain, but lacks the constant region. It may be an antibody fragment. Such an antibody fragment may be, for example, a diabody (Db), a single chain antibody, or sc (Fab ′) 2.
- Db diabody
- Fab ′ sc
- Db is a dimer composed of two polypeptide chains (Holliger P et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), EP404, 097, W093 / 11161, etc.)
- VL L chain variable region
- VH H chain variable region
- VL and VH encoded on the same polypeptide chain cannot form a single-chain variable region fragment due to the short linker between them, and form two antigen-binding sites by dimerization. .
- sc (Fv) 2 is a single-chain antibody in which four variable regions of two VLs and two VHs are connected by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231 (1- 2), 177-189).
- the two VHs and VLs can be derived from different monoclonal antibodies.
- the bispecific recognition (bispecific sc (Fv) 2) that recognizes two kinds of epitopes existing in the same antigen as disclosed in JournalJof Immunology (1994) 152 (11), 5368-5374 is also preferable.
- sc (Fv) 2 can be produced by methods known to those skilled in the art. For example, it can be prepared by linking scFv with a linker such as a peptide linker.
- the antigen-binding domain constituting sc (Fv) 2 is composed of two VHs and two VLs, VH, VL, VH, VL ([[ VH] Linker [VL] Linker [VH] Linker [VL]) are listed in this order, but the order of two VHs and two VLs is not particularly limited to the above configuration, They may be arranged in any order. For example, the following configuration can be given.
- sc (Fv) 2 The molecular form of sc (Fv) 2 is also described in detail in WO2006 / 132352. Based on these descriptions, those skilled in the art will appropriately use the molecular form of the sc (Fv) 2 for the production of the antigen-binding molecule disclosed herein. It is possible to produce a desired sc (Fv) 2.
- the antigen-binding molecule of the present invention may be conjugated with a carrier polymer such as PEG or an organic compound such as an anticancer agent. Moreover, a sugar chain addition sequence can be inserted, and a sugar chain can be suitably added for the purpose of obtaining a desired effect.
- a peptide linker is preferable.
- the length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art according to the purpose. However, the preferred length is 5 amino acids or more (the upper limit is not particularly limited, but usually 30 amino acids or less, preferably Is 20 amino acids or less), particularly preferably 15 amino acids.
- sc (Fv) 2 includes three peptide linkers, peptide linkers having the same length may be used, or peptide linkers having different lengths may be used.
- Synthetic chemical linkers are commonly used to crosslink peptides such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl) Suberate (BS3), dithiobis (succinimidyl propionate) (DSP), dithiobis (sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis (succinimidyl succinate) (EGS), ethylene Glycol bis (sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis [2- (succinimideoxycarbonyloxy ) Ethyl] sulfone (BSOCOES), bis [2- (sulfosuccinimidooxycarbonyloxy) ethyl] s
- F (ab ') 2 is two heavy chains containing two light chains and a constant region of the CH1 domain and a portion of the CH2 domain such that an interchain disulfide bond is formed between the two heavy chains including.
- F (ab ′) 2 constituting the polypeptide aggregate disclosed in the present specification is obtained by partially digesting a full-length monoclonal antibody or the like having a desired antigen-binding domain with a proteolytic enzyme such as pepsin. It can be suitably obtained by adsorbing on a protein A column and removing it.
- a proteolytic enzyme is not particularly limited as long as it can digest a full-length antibody so as to produce F (ab ′) 2 restrictively by appropriately setting the reaction conditions of the enzyme such as pH.
- pepsin and ficin can be exemplified.
- the antigen-binding molecule of the present invention can further contain additional modifications in addition to the amino acid modifications described above. Additional alterations can be selected from, for example, any of amino acid substitutions, deletions, modifications, or combinations thereof.
- the antigen-binding molecule of the present invention can be arbitrarily modified as long as it does not substantially change the target function of the molecule. For example, such mutations can be made by conservative substitution of amino acid residues.
- the modification gives a change to the target function of the antigen-binding molecule of the present invention, such a modification can be made as long as the change in the function is within the scope of the present invention. .
- the modification of the amino acid sequence in the present invention includes post-translational modification.
- post-translational modifications addition or deletion of sugar chains can be shown.
- the antigen-binding molecule of the present invention has an IgG1-type constant region
- the 297th amino acid residue of EU numbering can be modified with a sugar chain.
- the sugar chain structure to be modified is not limited.
- antibodies expressed in eukaryotic cells contain glycosylation in the constant region. Therefore, antibodies expressed in the following cells are usually modified with some sugar chain.
- Mammalian antibody-producing cells -Eukaryotic cells transformed with an expression vector containing DNA encoding the antibody.
- the eukaryotic cells shown here include yeast and animal cells.
- CHO cells and HEK293H cells are representative animal cells for transformation with an expression vector containing DNA encoding an antibody.
- those having no sugar chain modification at the position are also included in the antibody of the present invention.
- An antibody whose constant region is not modified with a sugar chain can be obtained by expressing a gene encoding the antibody in a prokaryotic cell such as Escherichia coli.
- a sialic acid may be added to the sugar chain of the Fc region (MAbs. 2010 2010 Sep-Oct; 2 (5): 519-27. .).
- the antigen-binding molecule of the present invention has an Fc region portion
- amino acid substitution J Immunol. 2006 Jan 1; 176 (1): 346-56, J Biol Chem. 2006 Aug 18 that improves the binding activity to FcRn
- 281 (33): 23514-24. Int Immunol. 2006 Dec; 18 (12): 1759-69., Nat Biotechnol. 2010 Feb; 28 (2): 157-9.
- WO / 2006/019447, WO / 2006/053301, WO / 2009/086320 amino acid substitution ((WO / 2009/041613)) for improving the heterogeneity and stability of the antibody may be added.
- antibody in the present invention is used in the broadest sense, and as long as the desired biological activity is exhibited, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody variants, antibody fragments, multiples Any antibody such as a specific antibody (eg, bispecific antibody), a chimeric antibody, a humanized antibody, etc. is included.
- the antibody of the present invention is not limited to the type of antigen, the origin of the antibody, etc., and may be any antibody.
- the origin of the antibody is not particularly limited, and examples thereof include a human antibody, a mouse antibody, a rat antibody, and a rabbit antibody.
- Humanized antibodies are also referred to as reshaped human antibodies.
- non-human animals for example, humanized antibodies obtained by grafting mouse antibody CDRs to human antibodies are known.
- General genetic recombination techniques for obtaining humanized antibodies are also known.
- Overlap-Extension-PCR is known as a method for transplanting mouse antibody CDRs into human FRs.
- FR amino acid residues can be substituted so that the CDR of the reshaped human antibody forms an appropriate antigen-binding site.
- amino acid sequence mutations can be introduced into FRs by applying the PCR method used for transplantation of mouse CDRs into human FRs.
- Transgenic animals having all repertoires of human antibody genes are used as immunized animals, and desired by DNA immunization. Human antibodies can be obtained.
- the V region of a human antibody is expressed as a single chain antibody (scFv) on the surface of the phage by the phage display method.
- Phages expressing scFv that bind to the antigen can be selected.
- the DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined.
- the V region sequence is fused in-frame with the sequence of the desired human antibody C region, and then inserted into an appropriate expression vector, whereby an expression vector can be prepared.
- the human antibody is obtained by introducing the expression vector into a suitable expression cell as described above and expressing the gene encoding the human antibody.
- These methods are already known (see International Publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, WO1995 / 015388).
- variable region constituting the antibody of the present invention can be a variable region that recognizes an arbitrary antigen.
- the “antigen” is not particularly limited and may be any antigen.
- antigens include 17-IA, 4-1 BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 Adenosine Receptor, A33, ACE, ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C, Activin RIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Addressins, adiponectin, ADP ribosyl cyclase-1, aFGF, AGE, ALCAM, ALK, ALK-1, ALK-7, allergen, alpha1
- HGF Hemopoietic growth factor
- Hep B gp120 Heparanase
- heparin cofactor II hepatic growth factor
- Bacillus anthracis protective antigen Hepatitis C virus E2 glycoprotein, Hepatitis E, Hepcidin, Her1, Her2 / neu ( ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HGF, HGFA, High molecular weight melanoma-associated antigen (HMW-MA A), HIV envelope proteins such as GP120, HIV MIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HMGB-1, HRG, Hrk, HSP47, Hsp90
- first antigen to which a variable region that can bind to two different antigens but cannot simultaneously bind to these antigens binds.
- second antigen include, for example, immune cell surface molecules (eg, T cell surface molecules, NK cell surface molecules, dendritic cell surface molecules, B cell surface molecules, NKT cell surface molecules, MDSC cell surface molecules Antigens (integrin, tissue factor, VEGFR, PDGFR, EGFR, IGFR, MET chemokine receptor, heparan) expressed in normal cells, tumor cells, tumor blood vessels, stromal cells, etc.
- immune cell surface molecules eg, T cell surface molecules, NK cell surface molecules, dendritic cell surface molecules, B cell surface molecules, NKT cell surface molecules, MDSC cell surface molecules
- Antigens integrin, tissue factor, VEGFR, PDGFR, EGFR, IGFR, MET chemokine receptor, heparan expressed in normal cells, tumor cells, tumor blood vessels, stromal cells, etc.
- Proteoglycan sulfate, CD44, fibronectin, DR5, TNFRSF, etc. are preferred, and the combination of “first antigen” and “second antigen” is any of the first antigen and the second antigen.
- first antigen and second antigen is any of the first antigen and the second antigen.
- the other antigen is preferably a molecule that is expressed on the surface of T cells or other immune cells.
- the combination of the “first antigen” and the “second antigen” includes any one of the first antigen and the second antigen expressed specifically on T cells, for example.
- the other antigen is a molecule that is expressed on immune cells and is different from the previously selected antigen.
- CD3 and a T cell receptor can be mentioned as molecules specifically expressed on T cells.
- CD3 is particularly preferable.
- the CD3 site to which the antigen-binding molecule of the present invention binds for example, in the case of human CD3, it binds to any epitope as long as it exists in the ⁇ chain, ⁇ chain or ⁇ chain sequence constituting human CD3 It may be a thing.
- an epitope present in the extracellular region of the ⁇ chain of the human CD3 complex is preferred.
- the structure of the ⁇ chain, ⁇ chain, or ⁇ chain constituting CD3 is such that the polynucleotide sequence is SEQ ID NO: 83 (NM_000073.2), 85 (NM_000732.4) and 87 (NM_000733.3).
- the sequences are described in SEQ ID NOs: 84 (NP_000064.1), 86 (NP_000723.1) and 88 (NP_000724.1) (in parentheses indicate RefSeq registration numbers).
- Still other antigens include Fc ⁇ receptor, TLR, lectin, IgA, immune checkpoint molecule, TNF superfamily molecule, TNFR superfamily molecule, and NK receptor molecule.
- first antigen and the “second antigen” to which the other variable region of the two variable regions of the antibody contained in the antigen-binding molecule of the present invention binds are different from the “third antigen”.
- antigens specific to tumor cells are preferable.
- antigens expressed as cells become malignant, abnormalities appearing on the cell surface or protein molecules when cells become cancerous.
- Sugar chains are also included.
- ALK receptor pleiotrophin receptor
- pleiotrophin pleiotrophin
- KS 1/4 pancreatic cancer antigen ovarian cancer antigen (CA125), prostatic acid phosphate, prostate specific antigen (PSA), Melanoma-associated antigen p97, melanoma antigen gp75, high molecular weight melanoma antigen (HMW-MAA), prostate specific membrane antigen, cancerous embryo antigen (CEA), polymorphic epithelial mucin antigen, human milk fat globule antigen, CEA, TAG-72 , CO17-1A, GICA 19-9, CTA-1 and LEA and other colorectal tumor associated antigens, Burkitt lymphoma antigen-38.13, CD19, human B lymphoma antigen-CD20, CD33, ganglioside GD2, ganglioside GD3, ganglioside GM2 and Tumor antigens induced by viruses such as melanoma-specific anti
- Differentiation antigens such as I antigen found in fetal erythrocytes, early endoderm I antigen found in adult erythrocytes, pre-implantation embryo, I (Ma) found in gastric cancer, M18, M39, bone marrow cells found in mammary epithelium SSEA-1, VEP8, VEP9, Myl, VIM-D5, D156-22 found in colorectal cancer, TRA-1-85 (blood group H), SCP-1 found in testis and ovarian cancer, colon C14 found in cancer, F3 found in lung cancer, AH6 found in stomach cancer, Y hapten, Ley found in embryonic cancer cells, TL5 (blood group A), EGF receptor found in A431 cells, E1 series found in pancreatic cancer (blood group B), FC10 found in embryonic cancer cells.
- Gastric cancer antigen CO-514 (blood group Lea) found in adenocarcinoma, NS-10, CO-43 (blood group Leb) found in adenocarcinoma, G49 found in EGF receptor of A431 cells, colon cancer MH2 (blood group ALeb / Ley), colon cancer 19.9, gastric cancer mucin, T5A7 found in bone marrow cells, R24 found in melanoma, 4.2 found in embryonic cancer cells, GD3, D1.1, OFA-1, GM2, OFA-2, GD2, and M1: 22: 25: 8 and SSEA-3 and SSEA-4, subcutaneous T-cell lymphoma antigen, MART-1 antigen found in 4-8 cell stage embryos, Sialyl Tn (STn) antigen, colon cancer antigen NY-CO-45, lung cancer antigen NY-LU-12 variant A, adenocarcinoma antigen ART1, tumor-associated brain-testis cancer antigen (cancer neuronal antigen MA2, tumor
- the antigen-binding molecule of the present invention can be produced by methods known to those skilled in the art.
- the antibody can be prepared by the following method, but is not limited thereto.
- Many combinations of host cells and expression vectors for producing antibodies by introducing a gene encoding an isolated polypeptide into a suitable host are known. Any of these expression systems can be applied to isolate the antigen-binding molecule of the present invention.
- animal cells, plant cells, or fungal cells can be used as appropriate. Specifically, the following cells can be exemplified as animal cells.
- Mammalian cells CHO (Chinese hamster ovary cell line), COS (Monkey kidney cell line), myeloma (Sp2 / O, NS0, etc.), BHK (baby hamster kidney cell line), HEK293 (human embryonic kidney cell line) with sheared adenovirus (Ad) 5 DNA), PER.C6 cell (human embryonic retinal cell line transformed with the Adenovirus Type 5 (Ad5) E1A and E1B genes), Hela, Vero, etc. (Current Protocols in Protein Science (May, 2001 , Unit 5.9, Table 5.9.1))
- Amphibian cells Xenopus oocytes, etc.
- Insect cells sf9, sf21, Tn5, etc.
- antibodies are used in Escherichia coli (mAbs 2012 Mar-Apr; 4 (2): 217-225.) And yeast (WO2000023579). ).
- the antibody produced in E. coli has no sugar chain added.
- sugar chains are added to antibodies produced in yeast.
- DNA encoding the heavy chain of an antibody the DNA encoding the heavy chain in which one or more amino acid residues in the variable region are substituted with other amino acids of interest, and the DNA encoding the light chain of the antibody To express.
- a DNA encoding a heavy chain or light chain in which one or more amino acid residues in the variable region are substituted with other amino acids of interest is, for example, an antibody prepared using a known method for an antigen. It can be obtained by obtaining a DNA encoding a variable region and appropriately introducing substitutions so that a codon encoding a specific amino acid in the region encodes another amino acid of interest.
- a DNA encoding a protein in which one or a plurality of amino acid residues in the variable region of an antibody prepared using a known method for an antigen is substituted with another amino acid of interest.
- the amino acid substitution site and the type of substitution are not particularly limited.
- Preferred regions for amino acid modification include regions exposed to solvent in the variable region and loop regions. Of these, CDR1, CDR2, CDR3, FR3 region and loop region are preferable.
- Kabat numbering 31 to 35, 50 to 65, 71 to 74, 95 to 102 of the heavy chain variable region, Kabat numbering 24 to 34, 50 to 56, 89 to 97 of the light chain variable region are preferable. More preferred are Kabat numbering 31, 52a-61, 71-74, 97-101 of the chain variable region, and Kabat numbering 24-34, 51-56, 89-96 of the L chain variable region.
- the amino acid modification is not limited to substitution, and may be any one of deletion, addition, insertion, modification, or a combination thereof.
- DNA encoding a heavy chain in which one or a plurality of amino acid residues in the variable region is substituted with another amino acid of interest can be produced by dividing it into partial DNAs.
- Examples of combinations of partial DNAs include DNA encoding a variable region and DNA encoding a constant region, or DNA encoding a Fab region and DNA encoding an Fc region, but are not limited to these combinations. is not.
- the DNA encoding the light chain can also be produced by dividing it into partial DNAs.
- DNA encoding a heavy chain variable region is incorporated into an expression vector together with DNA encoding a heavy chain constant region to construct a heavy chain expression vector.
- DNA encoding a light chain variable region is incorporated into an expression vector together with DNA encoding a light chain constant region to construct a light chain expression vector.
- the DNA encoding the target antibody When the DNA encoding the target antibody is incorporated into an expression vector, it is incorporated into the expression vector so that it is expressed under the control of an expression control region such as an enhancer or promoter. Next, host cells are transformed with this expression vector to express the antibody. In that case, a combination of an appropriate host and an expression vector can be used.
- vectors examples include M13 vectors, pUC vectors, pBR322, pBluescript, and pCR-Script.
- pGEM-T pDIRECT, pT7 and the like can be used in addition to the above vector.
- an expression vector is particularly useful.
- an expression vector for example, when the host is E. coli such as JM109, DH5 ⁇ , HB101, XL1-Blue, a promoter that can be efficiently expressed in E. coli, such as the lacZ promoter (Ward et al., Nature (1989) 341). , 544-546; FASEB J. (1992) 6, 2422-2427, incorporated herein by reference in its entirety, araB promoter (Better et al., Science (1988) 240, 1041-1043, in its entirety by reference) Are incorporated herein), or have a T7 promoter or the like.
- such vectors include pGEX-5X-1 (Pharmacia), “QIAexpress® system” (QIAGEN), pEGFP, or pET (in this case, the host expresses T7 RNA polymerase).
- pGEX-5X-1 Pulacia
- QIAexpress® system QIAGEN
- pEGFP pEGFP
- pET in this case, the host expresses T7 RNA polymerase.
- BL21 is preferred).
- the vector may also contain a signal sequence for polypeptide secretion.
- the signal sequence for polypeptide secretion is the pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4397, which is incorporated herein by reference in its entirety when produced in the periplasm of E. coli. Built in).
- Introduction of a vector into a host cell can be performed using, for example, the lipofectin method, the calcium phosphate method, or the DEAE-Dextran method.
- vectors for producing the polypeptide of the present invention include mammalian-derived expression vectors (for example, pcDNA3 (manufactured by Invitrogen), pEGF-BOS® (Nucleic® Acids.® Res.
- pEF Bacillus subtilis-derived expression vectors
- pCDM8 Bacillus subtilis-derived expression vectors
- insect cell-derived expression vectors eg “Bac-to-BAC baculovirus expression system” (GIBCO BRL), pBacPAK8)
- plant-derived expression vectors eg, pMH1, pMH2
- animal virus-derived expression vectors eg, pHSV, pMV, pAdexLcw
- retrovirus-derived expression vectors eg, pZIPneo
- yeast-derived expression vectors eg, “Pichia® Expression® Kit” (manufactured by Invitrogen), pNV11, SP-Q01
- Bacillus subtilis-derived expression vectors for example, pPL608, pKTH50.
- promoters required for expression in cells such as SV40 promoter (Mulligan et al., Nature (1979) 277, 108, incorporated herein by reference in its entirety), MMTV-LTR promoter, EF1 ⁇ promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322, incorporated herein in its entirety by reference), It is essential to have a CAG promoter (Gene.
- a CMV promoter etc.
- a gene for selecting transformed cells for example, More preferably, it has a drug resistance gene that can be discriminated by a drug (neomycin, G418, etc.).
- examples of such a vector include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.
- the EBNA1 protein is co-expressed for the purpose of increasing the copy number of the gene.
- a vector having the replication origin OriP is used. (Biotechnol Bioeng. 2001 Oct 20; 75 (2): 197-203., Biotechnol Bioeng. 2005 Sep 20; 91 (6): 670-7.)
- a vector having a DHFR gene complementary to the CHO cell lacking the nucleic acid synthesis pathway for example, , PCHOI, etc.
- amplifying with methotrexate (MTX) for example, COS with a gene expressing SV40 T antigen on the chromosome
- COS with a gene expressing SV40 T antigen on the chromosome An example is a method of transforming with a vector (such as pcD) having an SV40 replication origin using cells.
- a vector such as pcD
- the replication origin those derived from polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like can also be used.
- the expression vectors are selectable markers: aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase ( dhfr) gene and the like.
- APH aminoglycoside transferase
- TK thymidine kinase
- Ecogpt E. coli xanthine guanine phosphoribosyltransferase
- dhfr dihydrofolate reductase
- Antibody recovery can be performed, for example, by culturing transformed cells and then separating them from the inside of the cell or the culture solution of molecularly transformed cells.
- methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, C1q, FcRn, protein A, protein G column, affinity chromatography, ion exchange chromatography, gel filtration chromatography, etc. It can carry out in combination as appropriate.
- Knobs-into-holes technology (WO1996 / 027011, Ridgway JB et al., Protein Engineering (1996) 9 617-621, Merchant AM et al. Nature Biotechnology (1998) 16, 677-681) and a technique (WO2006 / 106905) that suppresses undesired association of H chains by introducing charge repulsion can be applied.
- the present invention provides a variable region of an antibody capable of binding to two different first antigens and second antigens, wherein the variable region does not bind to the first antigen and the second antigen simultaneously (first And a variable region that binds to a third antigen different from the first antigen and the second antigen (second variable region), comprising:
- a method for producing an antigen-binding molecule of the present invention comprising the step of preparing an antigen-binding molecule library having various amino acid sequences of the first variable region.
- a production method including the following steps can be mentioned: (i) an antigen-binding molecule in which at least one amino acid of a variable region of an antibody that binds to the first antigen or the second antigen is modified, wherein at least one of the amino acids of the modified variable region is different from each other Creating a library of antigen binding molecules comprising the region, (ii) an antigen comprising a variable region that has binding activity to the first antigen and the second antigen from the prepared library, but does not bind simultaneously with the first antigen and the second antigen; Selecting a binding molecule, (iii) culturing a host cell containing a nucleic acid encoding the variable region of the antigen-binding molecule selected in step (ii) and a nucleic acid encoding the variable region of the antigen-binding molecule that binds to the third antigen.
- variable region of an antibody that can bind to the first antigen and the second antigen but does not bind to the first antigen and the second antigen at the same time, and a variable region that binds to the third antigen.
- step of expressing an antigen binding molecule and (iv) recovering the antigen-binding molecule from the host cell culture.
- step (ii) may be the following selection step: (v) Among the prepared libraries, the first antigen and the second antigen have binding activity to the first antigen and the second antigen, but are expressed on different cells, respectively. Selecting an antigen-binding molecule comprising a variable region that does not bind.
- the antigen-binding molecule used in the step (i) is not particularly limited as long as it contains an antibody variable region, and may be an antibody fragment such as Fv, Fab, Fab ′, or an antibody containing an Fc region. There may be.
- amino acid to be modified for example, an amino acid that does not lose the binding to the antigen by amino acid modification is selected from the variable region of the antibody that binds to the first antigen or the second antigen.
- the amino acid modification of the present invention may be used alone or in combination.
- the number of combinations is not particularly limited. For example, 2 or more and 30 or less, preferably 2 or more and 25 or less, 2 or more and 22 or less, 2 or more and 20 or less, 2 or more 15 or less, 2 or more and 10 or less, 2 or more and 5 or less, 2 or more and 3 or less.
- the amino acid modification may be added only to the heavy chain variable region or the light chain variable region of the antibody, or may be appropriately distributed to both the heavy chain variable region and the light chain variable region.
- regions for amino acid modification include a region exposed to the solvent in the variable region and a loop region.
- CDR1, CDR2, CDR3, FR3 region and loop region are preferable.
- Kabat numbering 31 to 35, 50 to 65, 71 to 74, 95 to 102 of the heavy chain variable region, Kabat numbering 24 to 34, 50 to 56, 89 to 97 of the light chain variable region are preferable.
- More preferred are Kabat numbering 31, 52a-61, 71-74, 97-101 of the chain variable region, and Kabat numbering 24-34, 51-56, 89-96 of the L chain variable region.
- the amino acids in the above-mentioned regions of the variable region of the antibody that binds to the first antigen or the second antigen are randomly modified, or a desired antigen is previously prepared. It also includes inserting a peptide known to have binding activity to the above region. In this way, variable regions that can bind to the first antigen and the second antigen but cannot bind to these antigens at the same time are selected from the modified antigen-binding molecules. Thus, the antigen-binding molecule of the present invention can be obtained.
- Examples of peptides known to have a binding activity for a desired antigen in advance include the peptides shown in Table 1 above.
- variable region that can bind to the first antigen and the second antigen, but cannot bind to these antigens at the same time, and either the first antigen or the second antigen is a cell If both are present alone, both are present alone, or both are present on the same cell, they can bind to both the first and second antigens simultaneously. However, according to the above-mentioned method, it can be similarly confirmed whether or not the variable region cannot be bound simultaneously when expressed on different cells.
- the present invention provides a variable region of an antibody capable of binding to two different first antigens and second antigens, wherein the variable region does not bind to the first antigen and the second antigen simultaneously (first The antigen-binding molecule of the present invention, comprising the step of preparing an antigen-binding molecule library in which the amino acid sequence of the first variable region is diverse. Provide a way to do it.
- Examples of the method for producing such an antigen-binding molecule include a production method including the following steps: (i) an antigen-binding molecule in which at least one amino acid of a variable region of an antibody that binds to the first antigen or the second antigen is modified, wherein at least one of the amino acids of the modified variable region is different from each other Creating a library of antigen binding molecules comprising the region, (ii) an antigen comprising a variable region that has binding activity to the first antigen and the second antigen from the prepared library, but does not bind simultaneously with the first antigen and the second antigen; Selecting a binding molecule, (iii) A host cell containing a nucleic acid encoding the variable region of the antigen-binding molecule selected in step (ii) can be cultured to bind to the first and second antigens.
- a preferred region for the amino acid modification includes a heavy chain variable region. More preferably, a region exposed to the solvent in the variable region and a loop region are included. Of these, CDR1, CDR2, CDR3, FR3 region and loop region are preferable. Specifically, Kabat numbering 31 to 35, 50 to 65, 71 to 74, 95 to 102 of the heavy chain variable region, Kabat numbering 24 to 34, 50 to 56, 89 to 97 of the light chain variable region are preferable. More preferred are Kabat numbering 31, 52a-61, 71-74, 97-101 of the chain variable region, and Kabat numbering 24-34, 51-56, 89-96 of the L chain variable region.
- the step (ii) may be the following selection step: (v) Among the prepared libraries, the first antigen and the second antigen have binding activity to the first antigen and the second antigen, but are expressed on different cells, respectively. Selecting an antigen-binding molecule comprising a variable region that does not bind.
- the antigen-binding molecule used in the step (i) is not particularly limited as long as it contains an antibody variable region, and may be an antibody fragment such as Fv, Fab, Fab ′, or an antibody containing an Fc region. There may be.
- amino acid to be modified for example, an amino acid that does not lose the binding to the antigen by amino acid modification is selected from the variable region of the antibody that binds to the first antigen or the second antigen.
- the amino acid modification of the present invention may be used alone or in combination.
- the number of combinations is not particularly limited. For example, 2 or more and 30 or less, preferably 2 or more and 25 or less, 2 or more and 22 or less, 2 or more and 20 or less, 2 or more 15 or less, 2 or more and 10 or less, 2 or more and 5 or less, 2 or more and 3 or less.
- the amino acid modification may be added to either one of the heavy chain variable region or the light chain variable region of the antibody, or may be appropriately distributed and added to both the heavy chain variable region and the light chain variable region.
- the amino acids in the above-mentioned regions of the variable region of the antibody that binds to the first antigen or the second antigen are randomly modified, or a desired antigen is previously prepared. It also includes inserting a peptide known to have binding activity to the above region. In this way, variable regions that can bind to the first antigen and the second antigen but cannot bind to these antigens at the same time are selected from the modified antigen-binding molecules. Thus, the antigen-binding molecule of the present invention can be obtained.
- Examples of peptides known to have a binding activity for a desired antigen in advance include the peptides shown in Table 1 above.
- variable region that can bind to the first antigen and the second antigen, but cannot bind to these antigens at the same time, and either the first antigen or the second antigen is a cell Can bind to both the first antigen and the second antigen simultaneously if the other is present alone, if both are present alone, or if both are present on the same cell.
- antigen-binding molecules produced by the production method are also included in the present invention.
- the type and range of amino acid modification introduced by this method is not particularly limited.
- CD3 ( ⁇ chain, ⁇ chain or ⁇ chain constituting human CD3 in the case of human CD3) is selected as the first antigen, and CD3 and an optional second antigen And a library of antigen-binding molecules that bind to.
- library refers to a plurality of antigen-binding molecules or a plurality of fusion polypeptides containing antigen-binding molecules, or nucleic acids and polynucleotides encoding these sequences.
- sequences of a plurality of antigen-binding molecules or a plurality of fusion polypeptides comprising antigen-binding molecules contained in the library are not single sequences but are fusion polypeptides comprising antigen-binding molecules or antigen-binding molecules having different sequences from each other.
- a fusion polypeptide of the antigen-binding molecule of the present invention and a heterologous polypeptide can be produced.
- the fusion polypeptide is fused to at least a portion of a viral coat protein, such as a viral coat protein selected from the group consisting of pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, pVI and variants thereof. obtain.
- the antigen-binding molecules of the invention can be ScFv, Fab fragments, F (ab) 2 or F (ab ′) 2 , so in another embodiment, these antigen-binding molecules are heterologous.
- a library mainly comprising a plurality of fusion polypeptides which are fusion polypeptides with polypeptides and have different sequences from each other.
- these antigen-binding molecules and virus coat proteins such as pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, pVI and at least a part of a virus coat protein selected from the group consisting of variants thereof
- a library mainly composed of a plurality of fused polypeptides having different sequences from each other is provided.
- the antigen binding molecule of the present invention may further comprise a dimerization domain.
- the dimerization domain may be between the heavy or light chain variable region of an antibody and at least a portion of a viral coat protein.
- the dimerization domain can include a sequence comprising at least one of the dimerization sequences and / or one or more cysteine residues.
- This dimerization domain may preferably be linked to the C-terminus of the heavy chain variable region or constant region.
- a dimerization domain depending on whether the antibody variable region is made as a fusion polypeptide component with the viral coat protein component (no amber stop codon behind the dimerization domain), or , Depending on whether the antibody variable region is made primarily without the viral coat protein component (eg, having an amber stop codon after the dimerization domain) .
- bivalent display is provided by one or more disulfide bonds and / or a single dimerization sequence.
- the term “differing in sequence from each other” in the description of a plurality of antigen-binding molecules having different sequences means that the sequences of individual antigen-binding molecules in the library are different from each other. That is, the number of different sequences in the library reflects the number of independent clones having different sequences in the library, and is sometimes referred to as “library size”. In a normal phage display library, the number is 10 6 to 10 12 , and the library size can be increased to 10 14 by applying a known technique such as a ribosome display method. However, the actual number of phage particles used during phage library panning selection is typically 10 to 10,000 times larger than the library size.
- the term “different from each other” in the present invention means that the sequences of individual antigen-binding molecules in the library from which the number of library equivalents is excluded are different from each other, more specifically, antigen-binding molecules having different sequences from each other.
- the term “consisting mainly of” in the description of the library mainly composed of a plurality of antigen-binding molecules of the present invention means that the first and / or second antigens out of the number of independent clones having different sequences in the library. This reflects the number of antigen-binding molecules that differ in the binding activity of the antigen-binding molecule. Specifically, it is preferable that at least 10 4 antigen-binding molecules exhibiting such binding activity exist in the library. More preferably, the present invention provides a library in which at least 10 5 antigen-binding molecules exhibiting such binding activity are present. More preferably, the present invention provides a library in which at least 10 6 antigen-binding molecules exhibiting such binding activity are present.
- the present invention provides a library in which there are at least 10 7 antigen-binding molecules exhibiting such binding activity. Also preferably, the present invention provides a library in which at least 10 8 antigen-binding molecules exhibiting such binding activity are present.
- the ratio of antigen-binding molecules having different binding activities of the antigen-binding molecule to the first and / or second antigen can be suitably expressed. .
- the present invention provides that the antigen binding molecule exhibiting such binding activity is 0.1% to 80%, preferably 0.5% to 60%, more preferably 1% of the number of independent clones having different sequences in the library.
- a fusion polypeptide a polynucleotide molecule or a vector
- it can be expressed by the number of molecules or a ratio in the whole molecule as described above.
- a virus it can be expressed by the number of virus individuals or the ratio of the whole individual as described above.
- phage display is a technique for displaying a mutant polypeptide as a protein fused with at least a part of a coat protein on the surface of a phage, for example, a filamentous phage particle.
- the usefulness of phage display is the ability to rapidly and efficiently select sequences that bind with high affinity to a target antigen from a large library of randomized protein variants. Display of peptide and protein libraries on phage has been utilized to screen millions of polypeptides for specific binding properties. Multivalent phage display methods have been used to display small random peptides and proteins through fusion with gene III or gene VIII of filamentous phage (Wells and Lowman (Curr. Opin. Struct. Biol.
- phage display a library of proteins or peptides is fused to gene III or a portion thereof, and in the presence of wild type gene III protein so that the phage particles display 1 or 0 copies of the fusion protein. Expressed at low levels. Since the avidity effect is reduced compared to multivalent phage, selection is based on intrinsic ligand affinity and a phagemid vector is used, which simplifies DNA manipulation (Lowman and Wells , Methods: A Companion to Methods in Enzymology (1991) 3, 205-216).
- “Phagemid” is a plasmid vector having a bacterial origin of replication, eg, ColE1 and a copy of the intergenic region of bacteriophage.
- any known bacteriophage for example, filamentous bacteriophage and lambda type bacteriophage can be used as appropriate.
- the plasmid usually also contains a selectable marker for antibiotic resistance. DNA fragments cloned into these vectors can be propagated as plasmids.
- the plasmid replication mode changes to rolling circle replication, with one strand copy of plasmid DNA and packaged phage Generate particles.
- Phagemids can form infectious or non-infectious phage particles.
- the term includes a phagemid comprising a phage coat protein gene, or a fragment thereof, linked to a gene of this heterologous polypeptide as a gene fusion such that the heterologous polypeptide is displayed on the surface of the phage particle.
- phage vector means a double-stranded replicative form of a bacteriophage containing a heterologous gene and capable of replication.
- the phage vector has a phage origin of replication that allows phage replication and phage particle formation.
- the phage is preferably a filamentous bacteriophage such as M13, f1, fd, Pf3 phage or a derivative thereof, or a lambda type phage such as lambda, 21, phi80, phi81, 82, 424, 434, or the like or a derivative thereof.
- Oligonucleotides are known methods (eg, phosphotriester, phosphite, or phosphoramidite chemistry utilizing solid phase techniques such as those described in EP 266032, or Froeshler et al. (Nucl. Acids. Res. (1986) 14, 5399-5407), a short, single-stranded or double-stranded polydeoxynucleotide that is chemically synthesized by the method through a deoxynucleotide H-phosphonate intermediate). is there.
- Other methods include the polymerase chain reaction and other autoprimer methods described below, and oligonucleotide synthesis on a solid support. All of these methods are described in Engels et al.
- fusion protein and “fusion polypeptide” refer to a polypeptide having two parts covalently bonded to each other, each part being a polypeptide having different properties.
- This property can be a biological property such as in vitro or in vivo activity.
- This property can also be a single chemical or physical property, such as binding to the antigen of interest, catalysis of the reaction, and the like.
- the two portions can be linked directly by a single peptide bond or via a peptide linker containing one or more amino acid residues.
- the two parts and the linker are in the same reading frame.
- the two parts of the polypeptide are derived from heterologous or different polypeptides.
- coat protein refers to a protein in which at least part of the protein is present on the surface of the virus particle. From a functional point of view, a coat protein is any protein that binds to the viral particle during the process of virus construction in the host cell and remains associated with it until the virus infects other host cells.
- the coat protein can be a major coat protein or a minor coat protein.
- the minor coat protein is a coat protein normally present in the outer shell of the virus, preferably of at least about 5, more preferably at least about 7, more preferably at least about 10 or more proteins per virion. A copy exists.
- the major coat protein can be in the tens, hundreds or thousands of copies per virion.
- An example of a major coat protein is the p8 protein of filamentous phage.
- the following six methods are exemplified for preparing a library.
- 1. Inserting a peptide that binds to a second antigen (this term is used to include polypeptides and proteins) into an antigen-binding molecule that binds to the first antigen.
- a library in which various amino acids appear at a position where a loop in the antigen-binding molecule can be modified (extended) long can be prepared, and an antigen-binding molecule having binding activity against any second antigen can be obtained.
- an amino acid that maintains the binding activity with the first antigen is identified, 3.
- an antibody library in which various amino acids appear at a position where a loop in the antigen-binding molecule can be modified (extended) long is created, and binding to an arbitrary second antigen is performed.
- the glycosylation sequence (for example, NxS, NxT, where x is an amino acid other than P) is modified so that the sugar chain recognized by the sugar chain receptor is added (for example, a high-mannose sugar chain is added).
- high mannose type sugar chains can be obtained by adding kifunensine during antibody expression (MAbs. 2012 Jul-Aug; 4 (4): 475-87)) 6). 1.2.3.
- Cys, Lys, or a non-natural amino acid is inserted or substituted at a loop site or a site that could be modified to various amino acids, and a domain that binds to the second antigen is added by a covalent bond.
- Fab or a variable region part of the antigen-binding molecule is preferably used as the position where the amino acid in the antigen-binding molecule is substituted or the position where the peptide is inserted into the antigen-binding molecule.
- Preferred regions include regions exposed to solvent in the variable region and loop regions. Of these, CDR1, CDR2, CDR3, FR3 region and loop region are preferable.
- Kabat numbering 31 to 35, 50 to 65, 71 to 74, 95 to 102 of the heavy chain variable region, Kabat numbering 24 to 34, 50 to 56, 89 to 97 of the light chain variable region are preferable. More preferred are Kabat numbering 31, 52a-61, 71-74, 97-101 of the chain variable region, and Kabat numbering 24-34, 51-56, 89-96 of the L chain variable region.
- Method 1 As an embodiment of the method for inserting a peptide that binds to the second antigen into the antigen-binding molecule that binds to the first antigen described in Angew Chem Int Ed Engl. 2013 Aug 5; 52 (32): 8295- A method of inserting G-CSF as exemplified in 8 can also be mentioned.
- the peptide to be inserted can be obtained from a library displaying the peptide, but it is also possible to use the whole or part of a naturally occurring protein.
- CD3 in the case of human CD3, the ⁇ chain, ⁇ chain, or ⁇ chain that constitutes human CD3, it is considered to be involved in antigen binding. It is possible to make an amino acid modification at the site to produce a 1 amino acid modified antibody and judge. Methods known to those skilled in the art can be appropriately selected for evaluation of CD3 binding of 1 amino acid-modified antibody. For example, ELISA, FACS (fluorescence-activated cell-sorting), ALPHA screen (Amplified-Luminescent-Proximity-Homogeneous-Assay) and surface plasmon It can be measured by the BIACORE method using the resonance (SPR) phenomenon.
- SPR resonance
- Z (ratio of binding amount) is 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, preferably 0.8 or more, it can be considered that the binding to the antibody before modification is maintained. it can.
- Antibody libraries can be generated such that amino acids that maintain these bonds appear.
- ECM Extracellular matrix
- WO2012093704A1 Extracellular matrix
- ECM binding is evaluated according to the method of Reference Example 2, and the ECM binding value (ECL response; ECL reaction value) of each variant is determined as MRA (H chain sequence number). : 57, L chain SEQ ID NO: 58) divided by the antibody ECM binding value can be used.
- this value can be adopted as effective up to 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times.
- Antibody libraries can be generated so that amino acids selected in this way appear.
- a peptide in the library of the present invention, can be inserted into the variable region in order to enhance the diversity of the library.
- Preferred regions for peptide insertion include regions exposed to solvent in the variable region and loop regions.
- CDR1, CDR2, CDR3, FR3 region and loop region are preferable.
- Kabat numbering 31 to 35, 50 to 65, 71 to 74, 95 to 102 of the heavy chain variable region, Kabat numbering 24 to 34, 50 to 56, 89 to 97 of the light chain variable region are preferable. More preferred are Kabat numbering 31, 52a-61, 71-74, 97-101 of the chain variable region, and Kabat numbering 24-34, 51-56, 89-96 of the L chain variable region. More preferred is the region of Kabat numbering 99-100 of the heavy chain variable region.
- an amino acid that increases the binding activity with the antigen may be introduced together.
- the length of the inserted peptide is 1 to 3 amino acids, 4 to 6 amino acids, 7 to 9 amino acids, 10 to 12 amino acids, 13 to 15 amino acids, 15 to 20 amino acids, 21
- Examples include -25 amino acids, and preferably 1 to 3 amino acids, 4 to 6 amino acids, and 7 to 9 amino acids.
- the examination of the insertion position and length of the peptide for enhancing the diversity of the library can be carried out by preparing a molecule into which the peptide is inserted and evaluating the CD3 binding of the molecule.
- methods known to those skilled in the art can be selected as appropriate. For example, ELISA, FACS (fluorescence activated cell sorting), ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) or surface plasmon resonance (SPR) phenomenon is used. It can be measured by the BIACORE method.
- an antibody library for obtaining an antibody that binds to CD3 and a second antigen can be designed as follows. Step 1: Select amino acids that retain CD3 binding ability (CD3 binding amount should be 80% or more of unmodified antibody) For example, a library for obtaining an antibody that binds to CD3 and the second antigen can be prepared so that the amino acid selected in Step 1 appears.
- an antibody library for obtaining an antibody that binds to CD3 and a second antigen can be designed as follows.
- Step 1 Select amino acids that retain CD3 binding ability (CD3 binding amount should be 80% or more of unmodified antibody)
- Step 2 Insert amino acids between 99-100 (Kabat numbering) of heavy chain CDR3
- CD3 was enhanced by inserting amino acids into the CDR3 region in Step 2 in addition to Step 1.
- a library for obtaining antibodies that bind to the second antigen is provided as follows.
- an antibody library for obtaining an antibody that binds to CD3 and a second antigen can be designed as follows.
- Step 1 Select amino acids that retain CD3 binding ability (CD3 binding amount should be 80% or more of unmodified antibody)
- Step 2 Select amino acids whose ECM binding is within 10 times compared to MRA than before modification
- Step 3 Insert amino acids between 99-100 (Kabat numbering) of heavy chain CDR3
- amino acids that do not enhance ECM binding can be selected for amino acids that appear in the library, but the method is not limited thereto. Even if the library design does not go through step 2, it is possible to measure and evaluate ECM binding to an antigen-binding molecule obtained from the library.
- VH region CE115HA000 (SEQ ID NO: 52) is used as a template sequence of a CD3 (CD3 ⁇ ) -binding antibody
- amino acids used for library design are included in the heavy chain variable region. Kabat numbering 11th, 31st, 52a, 52b, 52b, 52c, 53, 54, 56, 57, 61, 72, 78, 98, 99, 100, 100a , 100b-position, 100c-position, 100d-position, 100e-position, 100f-position, 100g-position, 101-position, any one or more amino acids, etc. can be exemplified.
- VH region CE115HA000 (SEQ ID NO: 52) is modified with the amino acid modification of V11L / L78I is preferable, but is not limited thereto. Furthermore, the above-mentioned library in which the amino acid modification of V11L / D72A / L78I / D101Q is added to the VH region CE115HA000 (SEQ ID NO: 52) is preferable, but not limited thereto.
- VL region GLS3000 (SEQ ID NO: 53) is used as a template sequence of a CD3 (CD3 ⁇ ) -binding antibody
- amino acids used for library design are included in the light chain variable region. Kabat numbering 24th, 25th, 26th, 27th, 27a, 27b, 27b, 27c, 27e, 30th, 31st, 33rd, 34th, 51st, 52nd, 53rd, 54th , 55, 56, 74, 77, 89, 90, 92, 93, 94, 96, 107 may be exemplified.
- Designing a library in the present invention means, for example, NNK, TRIM Library, etc. (Gonzalez-Munoz A et al. MAbs 2012, Lee CV et al. J Mol Biol. 2004, Knappik A. et al. J Mol Biol. 2000, Tiller T et al. ⁇ MAbs 2013) using a known library technology, an antigen-binding domain in which an amino acid at a specific site is modified to a desired amino acid or a plurality of antigen-binding molecules containing an antigen-binding domain Designing a library containing the variants is included, but is not particularly limited to this embodiment.
- the “one or more amino acids” in the present invention are not particularly limited in the number of amino acids, and there are 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 It may be a kind of amino acid.
- the fusion polypeptide of the variable region of an antigen binding molecule can be presented in various ways on the surface of a cell, virus or phagemid particle. These embodiments include single chain Fv fragments (scFv), F (ab) fragments and multivalent forms of these fragments.
- the multivalent form is preferably a dimer of ScFv, Fab or F (ab ′), which are designated herein as (ScFv) 2, F (ab) 2 and F (ab ′) 2, respectively. Is done.
- One reason for the preference for multivalent forms is that multivalent forms can usually identify low-affinity clones or are more efficient for rare clones during the selection process. It is thought that this is a point having a plurality of antigen-binding sites that allow easy selection.
- nucleic acid sequences encoding the light chain variable region and heavy chain variable region of the antigen binding molecule are included in this vector.
- a nucleic acid sequence encoding the heavy chain variable region of an antigen binding molecule is fused to a viral coat protein component.
- the nucleic acid sequence encoding the light chain variable region of the antigen binding molecule is linked to the heavy chain variable region of the antigen binding molecule by a nucleic acid sequence encoding a peptide linker.
- Peptide linkers generally contain about 5 to 15 amino acids.
- sequences encoding a label useful for purification or detection for example, of the nucleic acid sequence encoding either the light chain variable region of the antigen binding molecule or the heavy chain variable region of the antigen binding molecule or both. Can be fused to the 3 'end.
- nucleic acid sequence encoding the variable region of the antigen-binding molecule and the constant region of the antigen-binding molecule is included in this vector.
- a nucleic acid encoding a light chain variable region is fused to a nucleic acid sequence encoding a light chain constant region.
- the nucleic acid sequence encoding the heavy chain variable region of the antigen binding molecule is fused to the nucleic acid sequence encoding the heavy chain constant CH1 region.
- nucleic acid sequences encoding heavy chain variable regions and constant regions are fused to nucleic acid sequences encoding all or part of a viral coat protein.
- the heavy chain variable region and constant region are preferably expressed as a fusion with at least a portion of the viral coat protein, and the light chain variable region and constant region are expressed separately from the heavy chain viral coat protein.
- the heavy and light chains bind to each other, but the bond can be covalent or non-covalent.
- the 3 ′ end of the nucleic acid sequence encoding the light chain constant region of the antigen binding molecule, or the heavy chain constant region of the antigen binding molecule, for example, where the other sequence encoding a polypeptide label useful for purification or detection is can be fused to either or both of the 3 ′ ends of the nucleic acid sequences encoding
- the vector constructed as described above is introduced into the host cell for amplification and / or expression.
- Vectors can be introduced into host cells by known transformation methods including electroporation, calcium phosphate precipitation, and the like. If the vector is an infectious particle such as a virus, the vector itself enters the host cell.
- the fusion protein is displayed on the surface of the phage particle by transfection of the host cell with a replicable expression vector into which the polynucleotide encoding the fusion protein has been inserted and production of the phage particle by known techniques.
- Replicable expression vectors can be introduced into host cells using a variety of methods.
- the vector can be introduced into the cells using electroporation methods as described in WO2000106717.
- Cells are optionally cultured in standard culture medium for approximately 6 to 48 hours (or until the OD at 600 nm is 0.6 to 0.8) at 37 ° C., and then the culture medium is centrifuged (eg, decantation). The culture supernatant is removed.
- the cell pellet is preferably resuspended in a buffer (eg 1.0 mM HEPES (pH 7.4)). The supernatant is then removed from the suspension by another centrifugation.
- a buffer eg 1.0 mM HEPES (pH 7.4)
- the resulting cell pellet is resuspended, for example, in glycerin diluted to 5-20% V / V.
- the cell pellet is obtained by removing the supernatant from the suspension again by centrifugation. Based on the measured cell concentration of the suspension obtained by resuspending the cell pellet in water or diluted glycerin, the final cell concentration is determined using water or diluted glycerin. To the desired concentration.
- preferable recipient cells include E. coli strain SS320 (Sidhu et al. (Methods Enzymol. (2000) 328, 333-363)) having electroporation response ability.
- E. coli strain SS320 was prepared by mating MC1061 cells with XL1-BLUE cells under conditions sufficient to transfer fertile episomes (F ′ plasmid) or XL1-BLUE to MC1061 cells. Deposit number 98795 is given to E. coli strain SS320 deposited at ATCC (10801 University Boulevard, Manassas, Virginia). Any F ′ episome that allows phage replication in this strain can be used in the present invention.
- Suitable episomes are available from strains deposited with the ATCC, or are commercially available (TG1, TG CJ236, CSH18, DHF ', ER2738, JM101, JM103, JM105, JM107, JM109, JM110, KS1000, XL1-BLUE, 71-18 etc.).
- the present invention provides a nucleic acid encoding the antigen-binding molecule of the present invention.
- the nucleic acid of the present invention may be in any form such as DNA or RNA.
- the present invention provides a vector containing the nucleic acid of the present invention.
- the type of vector can be appropriately selected by those skilled in the art depending on the host cell into which the vector is introduced. For example, the above-described vectors can be used.
- the present invention relates to a host cell transformed with the vector of the present invention.
- the host cell can be appropriately selected by those skilled in the art.
- the above-described host cell can be used.
- the present invention also provides a pharmaceutical composition comprising the antigen-binding molecule of the present invention and a medically acceptable carrier.
- the pharmaceutical composition of the present invention can be formulated by a known method by introducing a pharmaceutically acceptable carrier in addition to the antigen-binding molecule of the present invention. For example, it can be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable solution, or an injection of suspension.
- a pharmacologically acceptable carrier or medium specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative It is conceivable to prepare a pharmaceutical preparation by combining with a binder or the like as appropriate and mixing in a unit dosage form generally required for pharmaceutical practice.
- silicic acid lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride
- the carrier include polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, and inorganic salts. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained.
- a sterile composition for injection can be formulated in accordance with normal pharmaceutical practice using a vehicle such as distilled water for injection.
- Aqueous solutions for injection include, for example, isotonic solutions containing physiological saline, glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride, and suitable solubilizers such as You may use together with alcohol, specifically ethanol, polyalcohol, for example, propylene glycol, polyethylene glycol, nonionic surfactant, for example, polysorbate 80 (TM), HCO-50.
- alcohol specifically ethanol, polyalcohol, for example, propylene glycol, polyethylene glycol, nonionic surfactant, for example, polysorbate 80 (TM), HCO-50.
- oily liquid examples include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. Moreover, you may mix
- the prepared injection solution is usually filled into a suitable ampoule.
- ⁇ ⁇ Administration is preferably parenteral administration, and specific examples include injection, nasal administration, pulmonary administration, and transdermal administration. As an example of the injection form, it can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.
- the administration method can be appropriately selected depending on the age and symptoms of the patient.
- the dose of the pharmaceutical composition containing the polypeptide or the polynucleotide encoding the polypeptide can be selected, for example, in the range of 0.0001 mg / kg to 1000 mg / kg body weight at a time. Alternatively, for example, the dose can be selected in the range of 0.001 to 100,000 mg / body per patient, but is not necessarily limited to these values.
- the dose and administration method vary depending on the weight, age, symptoms, etc. of the patient, but can be appropriately selected by those skilled in the art.
- the present invention also includes a method for treating cancer, the antigen-binding molecule of the present invention for use in the treatment of cancer, and a method for producing a therapeutic agent for cancer, comprising the step of administering the antigen-binding molecule of the present invention.
- a process for producing a therapeutic agent for cancer comprising the use of an antigen binding molecule of the invention and a step of using the antigen binding molecule of the invention.
- an IgG-type antibody when it binds to an antigen in the variable region (Fab), it can bind to one molecule of Fc ⁇ R at the same time in the Fc region, so that cells expressing the antigen and Fc ⁇ R-expressing cells Cross-linking occurs.
- the antigen when the antigen is CD3, immune activation such as cytokine release occurs when T cells crosslink with Fc ⁇ R-expressing cells (J. Immunol. (1999) Aug 1, 163). (3), 1246-52).
- conventional multispecific antibodies bind to a plurality of antigens at the same time.
- the integrin ⁇ v ⁇ 3 known as an adhesion molecule
- the integrin ⁇ v ⁇ 3 is expressed in many cancer cells and blood vessels around the tumor, making it useful as a target molecule for targeting tumors (R. Haubner, PLoS Med., 2 , e70 (2005)), but on the other hand, it is also known to be expressed in various normal cells (Thromb Haemost. 1998 Nov; 80 (5): 726-34.). Therefore, if a multispecific antibody binds to both CD3 and integrin ⁇ v ⁇ 3 simultaneously, there is a possibility that normal cells may be damaged by the strong cytotoxic activity of T cells.
- variable (Fab) region a part of the variable (Fab) region binds to the first antigen and does not participate in the binding.
- a variable binding region (Dual Binding Fab) binding to the second antigen was considered (FIG. 1).
- FIG. 1 when two adjacent portions in one variable (Fab) region are essential for binding to each antigen, the binding of the second antigen occurs when the first antigen binds. Is inhibited, and similarly, when the binding of the second antigen is bound, the binding of the first antigen is inhibited.
- the antigen (third antigen) that binds to the other variable (Fab) region undergoes a crosslinking reaction with the first antigen (FIG. 4) and also with the second antigen. (FIG. 5).
- the constant region of the antibody an Fc region that binds to Fc ⁇ R can be used, or an Fc region with reduced binding activity to Fc ⁇ R can be used.
- an antibody having the action shown in FIG. 1 can be created if the following properties can be imparted by improving the variable (Fab) region to form a dual binding Fab.
- 1. It has binding activity for the first antigen. 2. It has binding activity for the second antigen. It does not bind to the first antigen and the second antigen at the same time.
- Does not bind to the first antigen and the second antigen at the same time means that the cell expressing the first antigen and the second antigen Do not cross-link two of the cells in which are expressed, or do not simultaneously bind to the first and second antigens expressed in separate cells, respectively, and If the antigen is not expressed on the cell membrane like a soluble protein, or both are present on the same cell, they can bind to both the first and second antigens simultaneously. However, when they are expressed on different cells, they may not be able to bind at the same time.
- variable (Fab) region by modifying the variable (Fab) region to form a dual binding Fab, if the following properties can be imparted, for example, an antibody having the action shown in FIG. 6 can be created. is there. 1. 1. has binding activity to the first antigen on T cells 2. It has binding activity for a second antigen on the antigen-presenting cell. Does not bind to the first and second antigens simultaneously
- Example 2 Preparation of anti-human, cynomolgus monkey CD3 ⁇ antibody CE115 (2-1) Preparation of hybridoma using human CD3, cynomolgus monkey CD3-expressing cell-immunized rat SD rat (female, 6 weeks old at the start of immunization, Charles River, Japan) ) Were immunized with human CD3 ⁇ or cynomolgus monkey CD3 ⁇ -expressing Ba / F3 cells as follows. When the first immunization was taken as day 0, 5 ⁇ 10 7 human CD3 ⁇ -expressing Ba / F3 cells were intraperitoneally administered on day 0 together with Freund's complete adjuvant (Difco).
- the fused cells were suspended in a semi-fluid medium (StemCells) to perform selective culture of the hybridoma and colonize the hybridoma.
- StemCells semi-fluid medium
- HAT selection medium (10% FBS / RPMI1640, 2 vol% HAT 50x concentrate (Dainippon Pharmaceutical), 5 vol% BM-Condimed H1 (Roche Diagnostics) ) was inoculated in a 96-well plate with 1 colony per well. After culturing for 3 to 4 days, the culture supernatant of each well was collected, and the rat IgG concentration in the culture supernatant was measured.
- the chimeric antibody H chain in which the rat antibody H chain variable region and the human antibody IgG1 chain constant region are combined, and the chimeric antibody L chain gene in which the rat antibody L chain variable region and the human antibody Kappa chain constant region are combined It was integrated into a cell expression vector.
- CE115 chimeric antibody was expressed and purified using the prepared expression vector (Reference Example 1).
- G1d with Gly and Lys removed from the C-terminus of IgG1, A5 with D356K and H435R mutations introduced into G1d, and B3 with K439E mutation introduced into G1d as the antibody H chain constant region, and Cetuximab-VH Cetuximab-VH-G1d (SEQ ID NO: 17), Cetuximab-VH-A5 (SEQ ID NO: 18), and Cetuximab-VH-B3 (SEQ ID NO: 19) were prepared according to the method of Reference Example 1. .
- the sequence corresponding to the H chain of the antibody having Cetuximab-VH in the variable region is shown as Cetuximab-VH-H1.
- D356K when an amino acid modification is indicated, it is indicated as D356K.
- the first alphabet (corresponding to D in D356K) means the alphabet when the amino acid residue before modification is shown in single letter notation, and the following number (corresponding to 356 in D356K) means the EU numbering of the modified part
- the last alphabet (corresponding to K in D356K) means the alphabet in the case where the amino acid residue after modification is indicated by a single letter.
- EGFR_ERY22_CE115 (FIG. 8) was prepared by replacing the VH domain and VL domain of Fab for EGFR. That is, by methods known to those skilled in the art such as PCR using a primer added with an appropriate sequence similar to the method described above, EGFR ERY22_Hk (SEQ ID NO: 20), EGFR ERY22_L (SEQ ID NO: 21), CE115_ERY22_Hh ( A series of expression vectors into which a polynucleotide encoding each of SEQ ID NO: 22) and CE115_ERY22_L (SEQ ID NO: 23) was inserted were prepared.
- Target molecule EGFR_ERY22_CE115 -Polypeptides encoded by polynucleotides inserted into expression vectors: EGFR_ERY22_Hk, EGFR_ERY22_L, CE115_ERY22_Hh, CE115_ERY22_L
- the mononuclear cell fraction layer was collected. After the cells of the mononuclear cell fraction were washed once with Dulbecco's Modified Eagle's Medium (SIGMA, 10% FBS / D-MEM) containing 10% FBS, the cell density was 4 ⁇ 10 6 / Prepared to 10 mL using 10% FBS / D-MEM. The cell solution thus prepared was used as a human PBMC solution in subsequent tests.
- SIGMA Dulbecco's Modified Eagle's Medium
- Cytotoxic activity was evaluated based on the cell growth inhibition rate using an xCELLigence real-time cell analyzer (Roche Diagnostics).
- the target cell the SK-pca13a cell line established by forcibly expressing human EGFR in the SK-HEP-1 cell line was used.
- SK-pca13a is detached from the dish, seeded at 100 ⁇ L / well on an E-Plate 96 plate (Roche Diagnostics) at 1 ⁇ 10 4 cells / well, and live cells using xCELLigence real-time cell analyzer Measurement was started.
- the plate was taken out from the xCELLigence real-time cell analyzer, and 50 ⁇ L of each antibody prepared at each concentration (0.004, 0.04, 0.4, 4 nM) was added to the plate. After reacting at room temperature for 15 minutes, add 50 ⁇ L (2 ⁇ 10 5 cells / well) of the human PBMC solution prepared in (2-5-1) and reset the plate in the xCELLigence real-time cell analyzer. Cell measurement was started. The reaction was carried out under conditions of 5% carbon dioxide gas and 37 ° C., and the cell growth inhibition rate (%) was determined from the Cell Index value 72 hours after the addition of human PBMC by the following formula.
- Cell growth inhibition rate (%) (AB) x 100 / (A-1)
- A shows the average Cell Index value in the wells to which no antibody is added (only target cells and human PBMC), and B shows the average Cell Index value in each well. The test was performed in triplicate.
- Dual binding Fab is a variable (Fab) region of CD3 (first antigen).
- target antigen second antigen
- second antigen but does not bind to CD3 (first antigen) and target antigen (second antigen) at the same time.
- amino acid modification is usually introduced into both the two H chains or L chains.
- the two Fabs of the antibody bind to the two antigens, respectively, so that the two Fabs and CD3 (first antigen) and the target antigen ( (Second antigen) may be simultaneously bound and cross-linked. Therefore, one Fab of the antibody is a Fab that binds to the third antigen or binds nothing, and the other Fab is a dual binding Fab, and CD3 (the first antigen) and the target antigen (the second antigen).
- Integrin ⁇ v ⁇ 3 known as an adhesion molecule, is expressed in many cancer cells and blood vessels around the tumor From the above, it is known that it is useful as a target molecule for targeting tumors, but is also expressed in various normal cells (Thromb Haemost. 1998 Nov; 80 (5): 726-34.). Therefore, it was considered that if CD3 and integrin ⁇ v ⁇ 3 are bound simultaneously, normal cells may be damaged by the strong cytotoxic activity of T cells.
- anti-EGFR antibody molecules could be targeted to tumor cells expressing integrin ⁇ v ⁇ 3 without damaging normal cells. That is, it binds to EGFR at one variable region (Fab), binds to CD3 as the first antigen, binds to integrin ⁇ v ⁇ 3 as the second antigen, and binds to CD3 and integrin ⁇ v ⁇ 3.
- Fab variable region
- Light chain variable region SEQ ID NO: 14 A heterodimerized antibody in which an RGD peptide was inserted into the heavy chain loop was prepared according to Reference Example 1. That is, a series of polynucleotides encoding any of the following together with polynucleotides encoding EGFR ERY22_Hk (SEQ ID NO: 20), EGFR ERY22_L (SEQ ID NO: 21) and CE115_ERY22_L (SEQ ID NO: 23), respectively.
- CE115_2 ERY22_Hh (SEQ ID NO: 24, Kabat numbering 52b-53 replaced with K and N, respectively), CE115_4 ERY22_Hh (SEQ ID NO: 25, Kabat numbering 52b-54 replaced with S and N, respectively), CE115_9 ERY22_Hh (SEQ ID NO: 26, RGD inserted between Kabat numbering 52a-52b), CE115_10 ERY22_Hh (SEQ ID NO: 27, RGD inserted between Kabat numbering 52b-52c), CE115_12 ERY22_Hh (SEQ ID NO: 28, RGD inserted between Kabat numbering 72-73), CE115_17 ERY22_Hh (SEQ ID NO: 29, replacing Kabat numbering 52b-52c with K and S, respectively), CE115_47 ERY22_Hh (SEQ ID NO: 30, RGD inserted between Kabat numbering 98-99), CE115_48 ERY22_Hh (SEQ ID NO: 31, insert RGD between Kabat number
- an antibody (EH240-Kn125 / EH240-Hl076 / L73) in which an RGD (Arg-Gly-Asp) peptide is inserted into the CH3 region of an antibody reported in J. Biotech, 155, 193-202, 2011 ; SEQ ID NO: 33/34/35) was prepared according to Reference Example 1.
- This molecule that binds to integrin ⁇ v ⁇ 3 through the CH3 region is thought to be able to bind to CD3 and integrin ⁇ v ⁇ 3 simultaneously.
- biotin-anti human IgG Ab (Southern biotech) diluted with a TBS solution containing 0.1% BSA and 0.1 g / L calcium chloride and 0.1 g / L magnesium chloride (denoted as a diluted (+) solution)
- Antibody solution prepared to 5 ⁇ g / mL or 1 ⁇ g / mL and integrin ⁇ v ⁇ 3 (R & D Systems) with sulfo-tag added to Nunc-Immuno (tm) MicroWell (tm) 96 well round plates (Nunc) 25 ⁇ L each was added to each well, mixed, and then incubated overnight at 4 ° C. to form antibody-antigen complexes.
- TBS solution referred to as blocking (+) solution
- BSA 0.1 g / L calcium chloride
- 0.1 g / L magnesium chloride 0.5% BSA
- MSD streptavidin plate
- the plate was washed 3 times with 250 ⁇ L of a TBS solution (denoted as TBS (+) solution) containing 0.1 g / L calcium chloride and 0.1 g / L magnesium chloride.
- 75 ⁇ L of the antibody-antigen complex solution was added to each well and incubated at room temperature for 2 hours to bind biotin-anti human IgG Ab to the streptavidin plate.
- the plate was washed 3 times with TBS (+) solution, 150 ⁇ L of READ buffer (MSD) was added to each well, and the sulfo-tag luminescence signal was detected with Sector Imager 2400 (MSD). .
- human CD3 ⁇ homodimeric protein with biotin diluted with diluted (+) solution, antibody solution prepared at 10 ⁇ g / mL or 5 ⁇ g / mL, and integrin with sulfo-tag added Add ⁇ v ⁇ 3 (R & D Systems) to each well of Nunc-Immuno (tm) MicroWell (tm) 96 well round plates (Nunc), mix, and incubate overnight at 4 ° C for antibody-antigen complex Formed. 150 ⁇ L of blocking (+) sputum solution was added to each well of streptavidin plate (MSD) and incubated overnight at 4 ° C.
- the plate was washed 3 times with 250 ⁇ L of a TBS (+) solution containing 0.1 g / L calcium chloride and 0.1 g / L magnesium chloride.
- 75 ⁇ L of the antibody-antigen complex solution was added to each well and incubated at room temperature for 2 hours to bind biotin-anti human IgG Ab to streptavidin plate.
- wash 3 times with TBS (+) solution add 150 ⁇ L of READ buffer (MSD) to each well, and detect the luminescence signal of sulfo-tag with Sector Imager 2400 (MSD). .
- a dual binding Fab molecule that has binding activity to any second antigen by inserting a peptide having binding activity to a protein as exemplified in WO2006036834 into a loop in the Fab.
- a peptide showing binding activity to a protein can be obtained by preparing a peptide library using a method known to those skilled in the art and selecting a peptide having a desired activity (Pasqualini R., Nature , 1996, 380 (6572): 364-6).
- a dual binding Fab molecule having binding activity to any second antigen Is considered possible. Since the variable region for the first antigen can be obtained by various methods known to those skilled in the art, by using such a library, any first antigen and any second antigen can be obtained. It can be said that it is possible to create a dual binding Fab molecule that has binding activity to and that cannot simultaneously bind to the first antigen and the second antigen.
- ERY22_Hk / EGFR ERY22_L / CE115_4 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_10 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_47 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_49 ERY22_Hh / CE115_ERY22_L is It was shown to bind to CD3 and integrin ⁇ v ⁇ 3 but not to CD3 and integrin ⁇ v ⁇ 3 simultaneously.
- TLR2 human toll-like receptor 2
- TLR2 which is known as a recognition receptor
- TLR2 is known to be expressed in normal cells other than immune cells such as epithelial cells and endothelial cells.
- the cancer antigen and TLR2 are simultaneously bound.
- immune cells that express TLR2 in the tumor environment can also be recruited and activated.
- Cancer cells injured by T cells can be activated by immune cells recruited by TLR2 and activated by processing antigens and presenting them to HLA, making T cells more active And may also induce acquired immunity.
- CD3 and TLR2 were simultaneously bound, it was considered that immune cells and normal cells might be damaged by the strong cytotoxic activity of T cells. Therefore, if a molecule that does not bind CD3 and TLR2 at the same time can be produced, it was considered that these cells could be recruited without damaging immune cells and normal cells that express TLR2.
- CE115 (heavy chain variable region SEQ ID NO: 13), which is a heterodimerized antibody in which one Fab is an EGFR binding domain and the other Fab is a CD3 binding domain and a TLR2 binding domain, is an antibody that binds to CD3 ⁇ .
- Light chain variable region SEQ ID NO: 14 A heterodimerized antibody antibody in which a TRL2-binding peptide was inserted into the heavy chain loop was prepared according to Reference Example 1.
- CE115_DU21 ERY22_Hh (SEQ ID NO: 37, TRL2-binding peptide inserted between Kabat numbering 52b-52c), CE115_DU22 ERY22_Hh (SEQ ID NO: 38, TRL2-binding peptide inserted between Kabat numbering 52b-52c), CE115_DU26 ERY22_Hh (SEQ ID NO: 39, TRL2-binding peptide inserted between Kabat numbering 72-73), CE115_DU27 ERY22_Hh (SEQ ID NO: 40, TRL2-binding peptide inserted between Kabat numbering 72-73).
- an antibody (CE115_ERY22_DU42_Hh, SEQ ID NO: 41) having a TLR2-binding peptide added to the C-terminus of the CH3 region, and an antibody (CE115_ ERY22_DU43_Hh, SEQ ID NO: 42) was prepared according to Reference Example 1. This molecule that binds to TLR2 via the CH3 region is thought to be able to bind to CD3 and TLR2 simultaneously.
- a dual binding Fab molecule that has binding activity to any second antigen by inserting a peptide having binding activity to a protein as exemplified in WO2006036834 into a loop in the Fab. Can be obtained.
- a peptide showing binding activity to a protein can be obtained by preparing a peptide library using a method known to those skilled in the art and selecting a peptide having a desired activity (Pasqualini R., Nature , 1996, 380 (6572): 364-6)).
- a dual binding Fab molecule having binding activity to any second antigen Is considered possible.
- variable region for the first antigen can be obtained by various methods known to those skilled in the art, by using such a library, any first antigen and any second antigen can be obtained. It can be said that it is possible to create a dual binding Fab molecule that has a binding activity to the first antigen and cannot simultaneously bind to the first antigen and the second antigen.
- Example 5 Modification of antibody for preparation of antibody that binds to CD3 and second antigen (5-1) Examination of insertion position and length of peptide capable of binding to second antigen Variable region on one side (Fab ) Binds to the cancer antigen, binds to the first antigen CD3 in the other variable region, binds to the second antigen, and does not bind to CD3 and the second antigen simultaneously.
- Fab Fab
- the antibody was produced according to Reference Example 1.
- Example 6 Library design for obtaining antibodies that bind to CD3 and second antigen (6-1) Antibody library for obtaining antibodies that bind to CD3 and second antigen (also known as Dual Fab Library) Call) The following six methods are exemplified as methods for selecting CD3 (CD3 ⁇ ) as the first antigen and obtaining an antibody that binds to CD3 (CD3 ⁇ ) and any second antigen. 1. A method of inserting a peptide or polypeptide that binds to the second antigen into the Fab domain that binds to the first antigen (in addition to the peptide insertion shown in Examples 3 and 4, Angew Chem Int Ed Engl.
- Peptides and polypeptides that bind can be obtained from libraries displaying peptides or polypeptides, but It is possible to use all or part of a naturally occurring protein. 2. As shown in Example 5, an antibody library in which various amino acids appear at a position where a loop in a Fab can be modified (extended) long and binding activity to an arbitrary second antigen is prepared. 2.
- a method for obtaining a Fab having an antibody from an antibody library using an antigen binding activity as an index Using an antibody created by site-directed mutagenesis from a Fab domain that is known to bind to CD3 in advance, the amino acids that maintain the binding activity to CD3 are identified, and the identified amino acids appear.
- the loop in the Fab is further modified to be longer
- the glycosylation sequence (for example, NxS, NxT, where x is an amino acid other than P) is modified so that the sugar chain recognized by the sugar chain receptor is added (for example, a high-mannose sugar chain is added).
- high mannose type sugar chains can be obtained by adding kifunensine during antibody expression (MAbs. 2012 Jul-Aug; 4 (4): 475-87))
- Cys, Lys, or an unnatural amino acid is inserted or substituted at a loop site or a site that could be modified to various amino acids, and binds to a second antigen.
- a method of covalently adding a domain (polypeptide, sugar chain, nucleic acid represented by a TLR agonist) (a method represented by an antibody drug conjugate, a method of covalently binding to Cys, Lys or an unnatural amino acid, mAbs 6: 1, 34-45; January / February 2014, WO2009 / 134891A2, Bioconjug Chem.
- a dual binding Fab that binds to the first antigen and the second antigen and does not bind to each other at the same time is obtained, and a domain that binds to any third antigen (referred to as the other variable region, (Described in Example 1) can be combined by methods known to those skilled in the art, such as common L chain, Cross mab, Fab arm exchange method.
- the constant region of the H chain is pE22Hh (L234A, L235A, N297A, D356C, T366S, L368A, Y407V is added to the sequence after CH1 of natural IgG1, and the GK sequence at the C terminus is deleted to obtain the DYKDDDDK sequence (sequence No .: 89) added sequence, SEQ ID NO: 54), and the K chain chain (SEQ ID NO: 55) was used as the L chain constant region.
- the modified sites are shown in Table 3.
- CD3 (CD3 ⁇ ) binding activity a 1 amino acid-modified antibody was obtained as a One arm antibody (an antibody lacking one Fab domain of natural IgG).
- the modified H chain is linked to the constant region pE22Hh and Kn010G3 (the amino acid sequence after the 216th amino acid sequence of natural IgG1 is modified with C220S, Y349C, T366W, H435R , SEQ ID NO: 56) and GLS3000 in which the kappa chain is linked to the 3 ′ side, and in the case of modification of the L chain, a sequence in which the Kappa chain is linked to the 3 ′ side of the modified L chain It was expressed and purified in FreeStyle293 cells using CE115HA000 and Kn010G3 in which pE22Hh was linked to the 3 ′ side as the H chain (the method of Reference Example 1 was used).
- Biacore T200 Evaluation Software (GE Healthcare) was used for calculation of each parameter.
- ECM Extracellular matrix binding evaluation of 1-amino acid-modified antibody
- ECM Extracellular matrix
- Example 5 Examination of peptide insertion location and length to enhance library diversity
- the peptide can be inserted without losing binding to CD3 (CD3 ⁇ ) using the GGS sequence at each location. It has been shown. If the loop can be extended in the Dual Fab Library, it will be a library that includes more types of molecules (also expressed as more diversity), and it will be possible to obtain Fab domains that bind to a variety of second antigens. It was thought to be. Therefore, since the binding activity was expected to decrease as the peptide was inserted, the V11L / D72A / L78I / D101Q modification was added to the CE115HA000 sequence so that the binding activity to CD3 ⁇ was increased.
- Example 5 a molecule into which a GGS linker was inserted was prepared, and CD3 binding was evaluated.
- the GGS sequence was inserted between 99-100 with Kabat numbering.
- the antibody molecule was expressed as a One arm antibody. Specifically, the above-mentioned H chain containing a GGS linker, Kn010G3 (SEQ ID NO: 56), and a GLS3000 (SEQ ID NO: 53) and Kappa sequence (SEQ ID NO: 55) linked as the L chain are used as a reference. Expression purification was performed according to Example 1.
- NNS base so that 6 amino acids are inserted between 99-100 (Kabat numbering) in CDR3 of the CE115HA340 sequence (SEQ ID NO: 59), which has higher CD3 ⁇ binding activity than CE115HA000, since a decrease in binding activity was expected.
- Primers were designed using The antibody molecule was expressed as a One arm antibody. Specifically, the above-mentioned H chain including the above-mentioned modification, Kn010G3 (SEQ ID NO: 56), and a sequence in which GLS3000 (SEQ ID NO: 53) and Kappa sequence (SEQ ID NO: 55) are linked as the L chain are employed. Expression purification was performed according to Reference Example 1. The obtained modified antibody was evaluated for binding by the method described in (6-3).
- Table 11 It has been clarified that the binding property to CD3 (CD3 ⁇ ) is maintained even when various amino acids appear in the extended amino acid site. Furthermore, Table 12 shows the results of evaluating whether or not non-specific binding is enhanced by the method shown in Reference Example 2. As a result, if a large number of amino acids having a positive charge in the side chain are contained in the extended loop of CDR3, the binding to ECM is enhanced, so there are amino acids having three or more positive charges in the side chain in the loop. It was hoped that it would not appear.
- the antigen binding site of Fab is diversified only by steps 1 and 3, it can be a library for identifying an antigen binding molecule that binds to the second antigen. Even for library designs that do not go through step 2, ECM binding can be measured and evaluated for the obtained molecules.
- the H chain of DualDFab Library is diversified as shown in Table 13 as the CDR of the sequence obtained by adding V11L / L78I mutation to FR (framework) of CE115HA000, and the CDR of GLS3000 is shown in Table 14 as L chain. Diversified.
- These antibody library fragments can be synthesized by DNA synthesis methods known to those skilled in the art.
- Dual Fab library (1) a library in which the H chain is diversified as shown in Table 13, and the L chain is fixed to the original sequence GLS3000 or the L chain with enhanced CD3 ⁇ binding described in Example 6; 2) A library in which the H chain is fixed to the original sequence (CE115HA000) or the H chain with enhanced CD3 ⁇ binding described in Example 6 and the L chain is diversified as shown in Table 14, (3) H A library can be created in which the chains are diversified as shown in Table 13 and the L chains are diversified as shown in Table 14.
- the H chain was entrusted to a DNA 2.0 DNA synthesis company with a library sequence diversified as shown in Table 13 as a CDR to a sequence obtained by adding the V11L / L78I mutation to CE115HA000 FR (framework). DNA fragment) was obtained.
- the obtained antibody library fragment was amplified by PCR and inserted into a phagemid for phage display. At this time, GLS3000 was selected as the L chain. Further, the constructed phagemid for phage display was introduced into E. coli by electroporation to produce E. coli having antibody library fragments.
- Example 7 Acquisition of Fab domain binding to CD3 and second antigen (IL6R) from Dual Fab Library (7-1) Acquisition of Fab domain binding to human IL6R Dual designed and constructed in Example 6 A Fab domain (antibody fragment) that binds to human IL6R was identified from the Fab library. Using biotin-labeled human IL6R as an antigen, antibody fragments capable of binding to human IL6R were concentrated. Phage production was performed from E. coli holding the constructed phagemid for phage display. A phage library solution was obtained by diluting a population of phage precipitated by adding 2.5 M NaCl / 10% PEG to the culture solution of Escherichia coli where phage production was performed, with TBS.
- IL6R dual antigen
- BSA was added to the phage library solution to a final concentration of 4% BSA.
- a panning method a panning method using an antigen immobilized on magnetic beads, which is a general method, was referred to (J. Immunol. Methods. (2008) 332 (1-2), 2-9, J. Immunol Methods. (2001) 247 (1-2), 191-203, Biotechnol. Prog. (2002) 18 (2) 212-20, Mol. Cell Proteomics (2003) 2 (2), 61-9).
- NeutrAvidin coated beads Sera-Mag SpeedBeads NeutrAvidin-coated
- Streptavidin coated beads Dynabeads M-280 Streptavidin
- the phage library solution was brought into contact with the antigen at room temperature for 60 minutes. Magnetic beads blocked with BSA were added, and the antigen-phage complex was allowed to bind to the magnetic beads for 15 minutes at room temperature. The beads were washed three times with TBST (TBS containing 0.1% Tween 20, TBS manufactured by TaKaRa), and then further washed twice with 1 mL of TBS. Thereafter, the beads to which 0.5 mL of 1 mg / mL trypsin had been added were suspended at room temperature for 15 minutes, and then the beads were immediately separated using a magnetic stand, and the phage solution was recovered.
- TST TBS containing 0.1% Tween 20, TBS manufactured by TaKaRa
- the recovered phage solution was added to 10 mL of E. coli strain ER2738 in the logarithmic growth phase (OD600 0.4-0.5).
- E. coli was infected with the phage by gently stirring the E. coli at 37 ° C. for 1 hour. Infected E. coli were seeded on 225 mm x 225 mm plates.
- a phage library solution was prepared by recovering the phages from the seeded E. coli culture solution. This cycle was called panning and repeated several times. In the second and subsequent panning, 40 pmol of biotin-labeled antigen was used. In the fourth panning, phages were concentrated using CD3 binding as an index.
- biotin-labeled CD3 ⁇ peptide antigen (amino acid sequence SEQ ID NO: 60)
- the phage library was brought into contact with the antigen at room temperature for 60 minutes. Magnetic beads blocked with BSA were added, and the antigen-phage complex was allowed to bind to the magnetic beads for 15 minutes at room temperature. The beads were washed with 1 mL of TBS containing 0.1% Tween20 and TBS. The beads to which 0.5 mL of 1 mg / mL trypsin had been added were suspended at room temperature for 15 minutes, and then the beads were immediately separated using a magnetic stand, and the phage solution was recovered.
- Phages recovered from the trypsinized phage solution were added to 10 mL of E. coli strain ER2738 in the logarithmic growth phase (OD600 0.4-0.7). E. coli was infected with the phage by gently stirring the E. coli at 37 ° C. for 1 hour. Infected E. coli were seeded on 225 mm x 225 mm plates. Next, the phage library solution was recovered by recovering the phage from the seeded E. coli culture solution. Furthermore, in order to prevent multiple phages from infecting a single E. coli, a phage library solution prepared from E. coli infected with the phages recovered by the fifth panning was again diluted with a 100,000-fold phage solution. To obtain a single colony.
- StreptaWell 96 microtiter plates (Roche) were coated with 100 ⁇ L of PBS containing biotinylated antigen (biotinylated CD3 ⁇ peptide or biotinylated human IL6R) at 4 ° C. overnight or at room temperature for 1 hour. Each well of the plate was washed with PBST to remove the antigen, and then the well was blocked with 250 ⁇ L of 4% BSA-TBS for 1 hour or longer. The plate containing the culture supernatant prepared in each well from which 4% BSA-TBS has been removed is allowed to stand at room temperature for 1 hour to allow the antibody displaying the phage to bind to the antigen present in each well. It was.
- biotinylated antigen biotinylated CD3 ⁇ peptide or biotinylated human IL6R
- CD3 ⁇ and the second antigen (human IL6R) can be evaluated. Furthermore, whether or not CD3 ⁇ and the second antigen (human IL6R) bind at the same time can be examined by the methods described in Examples 3 and 4 and the competition method. Competitive methods, for example, show that binding to CD3 ⁇ does not bind at the same time by reducing in the presence of a second antigen than with antibody alone.
- Example 8 Acquisition of Fab domain binding to CD3 and second antigen (human IgA) from Dual Fab Library (8-1) Acquisition of Fab domain binding to human IgA IgA is an abundant antibody in the body It is known as a molecule involved in biological defense in the intestine and mucosa, and is known to bind to Fc ⁇ R (Fc alpha Receptor) (J. Pathol. 208: 270 -282, 2006) .
- the Fab domain (antibody fragment) that binds to human IgA was identified from the Dual Fab library designed and constructed in Example 6. The antibody fragment capable of binding to human IgA was concentrated using biotin-labeled human IgA (described in Reference Example 3) as an antigen.
- Phage production was performed from E. coli holding the constructed phagemid for phage display.
- a phage library solution was obtained by diluting a population of phage precipitated by adding 2.5 M NaCl / 10% PEG to the culture solution of Escherichia coli where phage production was performed, with TBS.
- BSA was added to the phage library solution to a final concentration of 4% BSA.
- a panning method a panning method using an antigen immobilized on magnetic beads, which is a general method, was referred to (J. Immunol. Methods. (2008) 332 (1-2), 2-9, J. Immunol Methods. (2001) 247 (1-2), 191-203, Biotechnol. Prog.
- NeutrAvidin coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or Streptavidin coated beads (Dynabeads M-280 Streptavidin) were used as magnetic beads. Specifically, by adding 250 pmol of biotin-labeled antigen to the prepared phage library solution, the phage library solution was brought into contact with the antigen at room temperature for 60 minutes. Magnetic beads blocked with BSA were added, and the antigen-phage complex was allowed to bind to the magnetic beads for 15 minutes at room temperature.
- the beads were washed three times with TBST (TBS containing 0.1% Tween 20, TBS manufactured by TaKaRa), and then further washed twice with 1 mL of TBS. Thereafter, the beads to which 0.5 mL of 1 mg / mL trypsin had been added were suspended at room temperature for 15 minutes, and then the beads were immediately separated using a magnetic stand, and the phage solution was recovered. The recovered phage solution was added to 10 mL of E. coli strain ER2738 in the logarithmic growth phase (OD600 0.4-0.5). E. coli was infected with the phage by gently stirring the E. coli at 37 ° C. for 1 hour. Infected E.
- coli were seeded on 225 mm x 225 mm plates.
- a phage library solution was prepared by recovering the phages from the seeded E. coli culture solution. This cycle was called panning and was repeated four times.
- human IgA was 40 pmol.
- StreptaWell 96 microtiter plates (Roche) were coated with 100 ⁇ L PBS containing biotin-labeled antigen (biotin-labeled CD3 ⁇ peptide or biotin-labeled human IgA, Reference Example 3) at 4 ° C. overnight or at room temperature for 1 hour. Each well of the plate was washed with PBST to remove the antigen, and then the well was blocked with 250 ⁇ L of 0.1 ⁇ TBS / 150 mM NaCl / 0.02% Skim Milk for 1 hour or longer.
- biotin-labeled antigen biotin-labeled CD3 ⁇ peptide or biotin-labeled human IgA, Reference Example 3
- Whether or not the obtained antibody molecule having the Fab region binds to CD3 ⁇ and human IgA was determined by an electrochemiluminescence method (ECL method). Specifically, biotin-labeled CD3 ⁇ peptide (described in Example 7) or biotin-labeled human IgA (Reference Example 3) diluted with TBST solution (TaKaRa TBS plus 0.1% Tween20) and 2 ⁇ g An antibody solution prepared in 1 mL / mL and an anti-human IgG antibody (Invitrogen # 628400) with a sulfo-tag added to each well of Nunc-Immuno (tm) MicroWell (tm) 96 well round plates (Nunc) After each addition and mixing, the mixture was incubated at room temperature for 1 hour or more while protected from light to form an antibody-antigen complex.
- ECL method electrochemiluminescence method
- the Dual Fab Library is a library capable of obtaining a Fab domain having the ability to bind to the second antigen while retaining the ability to bind to CD3.
- TBST solution containing 0.5% BSA referred to as blocking solution; TBST solution with 0.1% Tween20 to TKaRa TBS
- MSD streptavidin plate
- the plate was washed 3 times with 250 ⁇ L of TBST solution. 50 ⁇ L each of the antibody-antigen complex solution was added to each well and incubated for 1 hour at room temperature to allow the biotinylated antigen-antibody-sulfo-tag antibody complex solution to bind to the streptavidin-plate via the biotinylated antigen.
- Dual Fab Library is a library that can acquire a Fab domain capable of binding to the second antigen while retaining the binding ability to CD3.
- Dual Fab Library in which only the H chain was diversified was used, but usually the larger the library size (also called diversity, meaning that the library includes various sequences), Since more antigen-binding molecules can be obtained, Dual Fab Library with diversified L chains can also be used for obtaining Dual Fab molecules in the same manner as shown in this example.
- Fab or antigen binding domain that binds to the third antigen can be obtained by a method known to those skilled in the art, for example, a binding antibody (or binding) from a hybridoma method or an antibody library.
- the antibody having an antigen-binding domain (for example, Fab) that binds to the identified third antigen and the Fab domain of the Dual Fab molecule can be identified using a multiple domain known to those skilled in the art. Multiplexed by specific antibody production methods, for example, the method of making antibodies with two L chains shared by sharing L chains (technology to control the interface of each domain of Fc region), CrossCMab method, Fab Arm Exchange method Specific antibodies can be obtained.
- a Dual Fab molecule can be identified, a desired multiplex can be obtained by combining the Fab that binds to the third antigen and the Dual Fab that binds to the first and second antigens shown in Example 8 by a method known to those skilled in the art. Specific antibodies can be obtained.
- Example 8 (5) CD3 / Human IgA Dual Fab Molecule
- a Dual Fab molecule that binds to CD3 ⁇ and human IgA and does not bind CD3 ⁇ and human IgA simultaneously is obtained.
- an antigen-binding domain that binds to the third antigen by a method known to those skilled in the art.
- IgA molecules modified to bind to EGFR one of the cancer antigens, were shown to induce cell death in EGFR-expressing cancer cells (J Immunol 2007; 179: 2936-2943 ).
- Fc ⁇ R an IgA receptor
- this dual Fab can be applied to cells expressing an arbitrary third antigen, to cells produced by cells expressing Fc ⁇ R via binding to CD3 ⁇ and T cells by binding to CD3 ⁇ and IgA. Both cytotoxic activities can be induced, and a strong cytotoxic activity can be expected.
- Example 9 Acquisition of Fab domain binding to CD3 and second antigen (human CD154) from Dual Fab Library (9-1) Acquisition of Fab domain binding to human CD154 Designed and constructed in Example 6 A Fab domain (antibody fragment) that binds to human CD154 was identified from the Dual Fab library. Using biotin-labeled human CD154 as an antigen, antibody fragments capable of binding to human CD154 were concentrated. Phage production was performed from E. coli holding the constructed phagemid for phage display. A phage library solution was obtained by diluting a population of phage precipitated by adding 2.5 M NaCl / 10% PEG to the culture solution of Escherichia coli where phage production was performed, with TBS.
- BSA was added to the phage library solution to a final concentration of 4% BSA.
- a panning method a panning method using an antigen immobilized on magnetic beads, which is a general method, was referred to (J. Immunol. Methods. (2008) 332 (1-2), 2-9, J. Immunol Methods. (2001) 247 (1-2), 191-203, Biotechnol. Prog. (2002) 18 (2) 212-20, Mol. Cell Proteomics (2003) 2 (2), 61-9).
- NeutrAvidin coated beads Sera-Mag SpeedBeads NeutrAvidin-coated
- Streptavidin coated beads Dynabeads M-280 Streptavidin
- the phage library solution was brought into contact with the antigen at room temperature for 60 minutes. Magnetic beads blocked with BSA were added, and the antigen-phage complex was allowed to bind to the magnetic beads for 15 minutes at room temperature. The beads were washed three times with TBST (TBS containing 0.1% Tween 20, TBS manufactured by TaKaRa), and then further washed twice with 1 mL of TBS. Thereafter, the beads to which 0.5 mL of 1 mg / mL trypsin had been added were suspended at room temperature for 15 minutes, and then the beads were immediately separated using a magnetic stand, and the phage solution was recovered.
- TST TBS containing 0.1% Tween 20, TBS manufactured by TaKaRa
- the recovered phage solution was added to 10 mL of E. coli strain ER2738 in the logarithmic growth phase (OD600 0.4-0.5).
- E. coli was infected with the phage by gently stirring the E. coli at 37 ° C. for 1 hour. Infected E. coli were seeded on 225 mm x 225 mm plates.
- a phage library solution was prepared by recovering the phages from the seeded E. coli culture solution. This cycle was called panning and was repeated 5 times. In the second and subsequent pannings, human CD154 was 40 pmol.
- ECL method electrochemiluminescence method (ECL method) whether or not antibody molecules having Fab regions that were bound to CD3 and human CD154 in (9-2) bind to CD3 and human CD154. .
- ECL method electrochemiluminescence method
- 25 ⁇ L of biotinylated CD3 or biotinylated human CD154 diluted with TBST solution, 25 ⁇ L of antibody solution prepared to 2 ⁇ g / mL, and 25 ⁇ L of anti-human IgG antibody (Invitrogen® # 628400) added with sulfo-tag was added to each well of Nunc-Immuno (tm) MicroWell (tm) 96 well round plates (Nunc), mixed, and incubated at room temperature for 1 hour or more while being shielded from light to form an antibody-antigen complex.
- TBST solution containing 0.5% BSA referred to as blocking solution; TBST solution with 0.1% Tween20 to TKaRa TBS
- MSD streptavidin plate
- the plate was washed 3 times with 250 ⁇ L of TBST solution. 50 ⁇ L each of the antibody-antigen complex solution was added to each well and incubated for 1 hour at room temperature to allow the biotinylated antigen-antibody-sulfo-tag antibody complex solution to bind to the streptavidin-plate via the biotinylated antigen.
- the Dual Fab Library is a library capable of obtaining a Fab domain having the ability to bind to the second antigen while retaining the ability to bind to CD3.
- biotinylated human CD154 25 ⁇ L diluted with TBST solution, antibody solution 12.5 ⁇ L prepared to 1 ⁇ g / mL, TBST or CD3 ⁇ homodimeric protein (9.4 pmol / ⁇ L) 12.5 for competition Add ⁇ L and 25 ⁇ L of anti-human IgG antibody added with sulfo-tag (Invitrogen) # 628400) to each well of Nunc-Immuno (tm) MicroWell (tm) 96 well round plates (Nunc). The mixture was incubated at room temperature for 1 hour or longer to form an antibody-antigen complex.
- TBST solution containing 0.5% BSA referred to as blocking solution; TBST solution with 0.1% Tween20 to TKaRa TBS
- MSD streptavidin plate
- the plate was washed 3 times with 250 ⁇ L of TBST solution. 50 ⁇ L each of the antibody-antigen complex solution was added to each well and incubated for 1 hour at room temperature to allow the biotinylated antigen-antibody-sulfo-tag antibody complex solution to bind to the streptavidin-plate via the biotinylated antigen.
- Dual Fab Library is a library that can obtain a Fab domain having the ability to bind to the second antigen while retaining the ability to bind CD3.
- Dual Fab Library in which only the H chain was diversified was used, but usually the larger the library size (also called diversity, meaning that the library includes various sequences), Since more antigen-binding molecules can be obtained, Dual Fab Library with diversified L chains can also be used for obtaining Dual Fab molecules in the same manner as shown in this example.
- Fab or antigen binding domain that binds to the third antigen can be obtained by methods known to those skilled in the art, for example, binding antibodies or antigen binding from hybridoma methods or antibody libraries.
- the antibody having an antigen-binding domain (for example, Fab) that binds to the identified third antigen and the Fab domain of the Dual Fab molecule can be identified using a domain selection method. Multispecificity by the production method of sex antibody, for example, the method of making an antibody with two different H chains by sharing L chain (technology to control the interface of each domain of Fc region), Cross Mab method, Fab Arm Exchange method Sex antibodies can be obtained.
- a Dual Fab molecule can be identified, a desired multiplex can be obtained by combining the Fab that binds to the third antigen and the Dual Fab that binds to the first and second antigens shown in Example 9 by a method known to those skilled in the art.
- Specific antibodies can be obtained. From the above examples, it was shown that a molecule that binds to CD3 ⁇ and the second antigen can be obtained by adapting Dual Fab library to many types of antigens. Further, in Examples 8 and 9, the first antigen ( It was revealed that molecules that bind to CD3 ⁇ ) and the second antigen but do not bind to the first antigen and the second antigen simultaneously can be obtained. As described above, it is possible to identify a Fab that binds to the third antigen by a method known to those skilled in the art. Therefore, the desired antibody described in Example 1 can be obtained by using the Dual Fab library. it can.
- Example 9 (9-5) CD3 / Human CD154 Dual Fab Molecule
- a Dual Fab molecule that binds to CD3 ⁇ and human CD154 and does not bind CD3 ⁇ and human CD154 at the same time is obtained.
- an antigen-binding domain that binds to the third antigen by a method known to those skilled in the art.
- an agonist antibody of CD40 which is a receptor for CD154, enhances antitumor activity in a method of transferring cancer antigen-reactive T cells (J Immunother. 2012 Apr; 35 (3): 276-82 .).
- a dual Fab molecule that binds to CD3 and CD154 can be constructed.
- an antibody exhibiting agonist activity against CD40 is selected via CD154, antitumor effect via CD40 is expected. It can. That is, this dual Fab is capable of inhibiting the cytotoxic activity of T cells by binding to CD3 ⁇ and the antitumor effect of CD40 agonistic signals through binding to CD154 on cells expressing an arbitrary third antigen. An increase can be expected.
- the antibody was purified from the obtained culture supernatant by a method known to those skilled in the art using rProtein A Sepharose TM Fast Flow (GE Healthcare).
- the purified antibody concentration was determined by measuring the absorbance at 280 nm using a spectrophotometer, and calculating the antibody concentration using the extinction coefficient calculated by the PACE method from the obtained value (Protein Science 1995; 4: 2411- 2423).
- ECM-immobilized plate add 150 ⁇ L of ECL Blocking Buffer (PBS with 0.5% BSA and 0.05% Tween 20) to each well, and allow to stand at room temperature for at least 2 hours or at 4 ° C. Let stand overnight.
- the antibody sample was diluted to 9 ⁇ g / mL using PBS-T (0.05% Tween 20 added to PBS). Dilute the secondary antibody to 2 ⁇ g / mL with ECLDB (PBS plus 0.1% BSA and 0.01% Tween 20), and add 20 ⁇ L of antibody solution to the round-bottom plate in which 10 ⁇ L of ECLDB is dispensed into each well.
- a gene fragment encoding a protein (SEQ ID NO: 80) in which human IgA-Fc and AviTag sequences are linked is incorporated into an animal cell expression vector, and the constructed plasmid vector is used in FreeStyle293 cells (Invitrogen) using 293Fectin (Invitrogen). Introduced. At this time, a gene expressing EBNA1 (SEQ ID NO: 81) and a gene expressing biotin ligase (BirA, SEQ ID NO: 82) were simultaneously introduced, and biotin was added for the purpose of biotin labeling human IgA-Fc. Cells into which the gene had been introduced were cultured for 6 days at 37 ° C.
- the cell culture solution containing the target human IgA-Fc was filtered through a 0.22 ⁇ m bottle top filter to obtain a culture supernatant. Apply the culture supernatant diluted with the same solution to HiTrap Q HP (GE Healthcare) equilibrated with 20 mM Tris-HCl, pH 7.4, and elute the target human IgA-Fc with NaCl concentration gradient I let you.
- HiTrap Q HP GE Healthcare
- the HiTrap Q HP eluate diluted with the same solution was applied to a SoftLink Avidin column (Promega) equilibrated with 50 mM Tris-HCl, pH 8.0, and 5 mM biotin, 150 mM NaCl, 50
- the target human IgA-Fc was eluted with mM Tris-HCl, pH 8.0.
- purified humans were removed by gel filtration chromatography using Superdex200 (GE Healthcare) to remove aggregates, which were impurities of no interest, and the buffer was replaced with 20 mM Histidine-HCl, 150 mM NaCl, pH 6.0. IgA-Fc was obtained.
- the present invention makes it possible to enhance the activity produced by antigen-binding molecules and avoid cross-linking between different cells caused by binding to antigens expressed on different cells, which are thought to cause side effects.
- a polypeptide suitable as a pharmaceutical product is provided.
Abstract
Description
また、従来技術のBispecific抗体では、一つ目の抗原であるがん抗原(EpCAM)と二つ目の抗原であるCD3εの両方の抗原がFcγRと同時に結合し得るため、FcγRと2つ目の抗原のCD3εの同時結合によるこのような副作用を回避することは分子構造的に不可能である。 However, since trifunctional antibody binds CD3ε and FcγR simultaneously even in the absence of cancer antigen, CD3ε-expressing T cells and FcγR-expressing cells are cross-linked even in the absence of cancer cells. And various cytokines are produced in large quantities. Due to the induction of production of various cytokines independent of cancer antigens, the administration of trifunctional antibodies is currently limited to the abdominal cavity (Cancer Treat Rev. 2010 Oct 36 (6), 458-67 (non-patent literature) 16)), systemic administration is extremely difficult due to serious cytokine storm-like side effects (Cancer Immunol Immunother. 2007 Sep; 56 (9): 1397-406 (Non-patent Document 18)).
In addition, in the bispecific antibody of the prior art, both the antigen of cancer antigen (EpCAM) as the first antigen and the antigen of CD3ε as the second antigen can bind simultaneously with FcγR. It is molecularly impossible to avoid such side effects due to simultaneous binding of antigen CD3ε.
しかしながら、このような抗体でも、分子構造的に、がん抗原に結合しつつCD3εとFcγRの2つの免疫レセプターに作用することはできない。
これまで、副作用を回避しつつ、癌抗原特異的に、T細胞による細胞障害活性とT細胞以外の細胞による細胞傷害活性の両方を作用させることのできる抗体は知られていない。 In recent years, there has been provided an improved antibody that causes cytotoxic activity by T cells while avoiding side effects by using an Fc region with reduced binding activity to FcγR (WO2012 / 073985).
However, even such an antibody cannot act on two immune receptors, CD3ε and FcγR, while binding to a cancer antigen in terms of molecular structure.
Until now, no antibody has been known that can cause both cytotoxic activity by T cells and cytotoxic activity by cells other than T cells in a cancer antigen-specific manner while avoiding side effects.
〔1〕第1の抗原および該第1の抗原とは異なる第2の抗原に結合することができるが、第1の抗原と第2の抗原に同時には結合しない、抗体の可変領域、並びに
該第1の抗原および第2の抗原とは異なる第3の抗原に結合する可変領域
を含む、抗原結合分子。
〔2〕第1の抗原および該第1の抗原とは異なる第2の抗原に結合することができるが、第1の抗原と第2の抗原に同時には結合しないように、重鎖可変領域のアミノ酸が改変された抗体の可変領域を含む、抗原結合分子。
〔3〕第1の抗原と第2の抗原に同時には結合しない可変領域が、それぞれ異なる細胞上で発現している第1の抗原と第2の抗原に同時には結合しない可変領域である、〔1〕又は〔2〕に記載の抗原結合分子。
〔4〕さらに、抗体のFc領域を含む、〔1〕から〔3〕のいずれかに記載の抗原結合分子。
〔5〕Fc領域のFcγRに対する結合活性が、天然型ヒトIgG1抗体のFc領域と比較して低下しているFc領域である、〔4〕に記載の抗原結合分子。
〔6〕多重特異性抗体である、〔1〕から〔5〕のいずれかに記載の抗原結合分子。
〔7〕第1の抗原と第2の抗原に結合することができる抗体の可変領域が、少なくとも1つのアミノ酸の改変が導入されている可変領域である、〔1〕から〔6〕のいずれかに記載の抗原結合分子。
〔8〕前記改変が、少なくとも1つのアミノ酸の置換又は挿入である、〔7〕に記載の抗原結合分子。
〔9〕前記改変が、第1の抗原に結合する可変領域のアミノ酸配列の一部の、第2の抗原に結合するアミノ酸配列への置換、又は、第1の抗原に結合する可変領域のアミノ酸配列への、第2の抗原に結合するアミノ酸配列の挿入である、〔7〕又は〔8〕に記載の抗原結合分子。
〔10〕挿入されるアミノ酸の数が1~25個である、〔8〕又は〔9〕に記載の抗原結合分子。
〔11〕改変されるアミノ酸が、抗体の可変領域のCDR1、CDR2、CDR3又はFR3領域のアミノ酸である、〔7〕から〔10〕のいずれかに記載の抗原結合分子。
〔12〕改変されるアミノ酸が、ループ領域のアミノ酸である、〔7〕から〔11〕のいずかに記載の抗原結合分子。
〔13〕改変されるアミノ酸が、抗体のH鎖可変領域のKabatナンバリング31~35、50~65、71~74及び95~102、L鎖可変領域のKabatナンバリング24~34、50~56及び89~97から選ばれる少なくとも1つのアミノ酸である、〔7〕から〔11〕のいずれかに記載の抗原結合分子。
〔14〕第1の抗原又は第2の抗原のいずれか1つが、T細胞表面に特異的に発現している分子であり、他方の抗原が、T細胞又は他の免疫細胞表面に発現している分子である、〔1〕から〔13〕のいずれかに記載の抗原結合分子。
〔15〕第1の抗原又は第2の抗原のいずれか1つがCD3であり、他方の抗原がFcγR、TLR、レクチン、IgA、免疫チェックポイント分子、TNFスーパーファミリー分子、TNFRスーパーファミリー分子又はNKレセプター分子である、〔14〕に記載の抗原結合分子。
〔16〕第3の抗原が、がん組織特異的に発現している分子である、〔14〕又は〔15〕に記載の抗原結合分子。
〔17〕〔1〕から〔16〕のいずれかに記載の抗原結合分子及び医学的に許容し得る担体を含む、医薬組成物。
〔18〕〔1〕から〔16〕のいずれかに記載の抗原結合分子を製造する方法であって、工程(i)~(iv)を含む方法:
(i)第1の抗原又は第2の抗原に結合する抗体の可変領域の少なくとも1つのアミノ酸が改変された抗原結合分子であって、該改変された可変領域のアミノ酸の少なくとも1つが互いに異なる可変領域を含む抗原結合分子のライブラリーを作製する工程、
(ii)作製されたライブラリーの中から、第1の抗原及び第2の抗原に対して結合活性を有するが、該第1の抗原及び第2の抗原と同時には結合しない可変領域を含む抗原結合分子を選択する工程、
(iii)工程(ii)で選択された抗原結合分子の該可変領域をコードする核酸、及び/または、第3の抗原に結合する抗原結合分子の可変領域をコードする核酸とを含む宿主細胞を培養して、第1の抗原と第2の抗原に結合することができるが該第1の抗原と第2の抗原とが同時には結合しない抗体の可変領域、及び/または、第3の抗原に結合する可変領域を含む、抗原結合分子を発現させる工程、並びに
(iv)前記宿主細胞培養物から抗原結合分子を回収する工程。
〔19〕工程(ii)において選択する抗原結合分子に含まれる、第1の抗原と第2の抗原に同時には結合しない可変領域が、それぞれ異なる細胞上で発現している第1の抗原と第2の抗原に同時には結合しない可変領域である、〔18〕に記載の製造方法。
〔20〕工程(iii)において培養する宿主細胞が、抗体のFc領域をコードする核酸をさらに含む、〔18〕又は〔19〕に記載の製造方法。
〔21〕Fc領域のFcγRに対する結合活性が、天然型ヒトIgG1抗体のFc領域と比較して低下しているFc領域である、〔20〕に記載の製造方法。
〔22〕製造する抗原結合分子が多重特異性抗体である、〔18〕から〔21〕のいずれかに記載の製造方法。
〔23〕工程(i)における、可変領域の少なくとも1つの改変されたアミノ酸が、置換又は挿入されたアミノ酸である、〔18〕から〔22〕のいずれかに記載の製造方法。
〔24〕挿入されたアミノ酸の数が1~25個である、〔23〕に記載の製造方法。
〔25〕改変が、抗体の可変領域のCDR1、CDR2、CDR3又はFR3領域のアミノ酸の改変である、〔18〕から〔24〕のいずれかに記載の製造方法。
〔26〕改変が、ループ領域のアミノ酸の改変である、〔18〕から〔25〕のいずれかに記載の製造方法。
〔27〕改変が、抗体のH鎖可変領域のKabatナンバリング31~35、50~65、71~74及び95~102、L鎖可変領域のKabatナンバリング24~34、50~56及び89~97から選ばれる少なくとも1つのアミノ酸の改変である、〔18〕から〔25〕のいずれかに記載の製造方法。
〔28〕第1の抗原又は第2の抗原のいずれか1つが、T細胞表面に特異的に発現している分子であり、他方の抗原が、T細胞又は他の免疫細胞表面に発現している分子である、〔18〕から〔27〕のいずれかに記載の製造方法。
〔29〕第1の抗原又は第2の抗原のいずれか1つがCD3であり、他方の抗原がFcγR、TLR、IgA、レクチン、免疫チェックポイント分子、TNFスーパーファミリー分子、TNFRスーパーファミリー分子又はNKレセプター分子である、〔28〕に記載の製造方法。
〔30〕第3の抗原が、がん組織特異的に発現している分子である、〔28〕又は〔29〕に記載の製造方法。
〔31〕〔1〕から〔16〕のいずれかに記載の抗原結合分子を投与する工程を含む、がんの治療方法。
〔32〕がんの治療において使用するための、〔1〕から〔16〕のいずれかに記載の抗原結合分子。
〔33〕がんの治療剤の製造における、〔1〕から〔16〕のいずれかに記載の抗原結合分子の使用。
〔34〕〔1〕から〔16〕のいずれかに記載の抗原結合分子を使用する工程を含む、がんの治療剤を製造するためのプロセス。
〔35〕上記に記載の1又は複数の態様を任意に組み合わせたものも、当業者の技術常識に基づいて技術的に矛盾しない限り、本発明に含まれることが当業者には当然に理解される。 More specifically, the present invention relates to the following.
[1] a variable region of an antibody that can bind to a first antigen and a second antigen different from the first antigen, but does not bind to the first antigen and the second antigen simultaneously, and An antigen binding molecule comprising a variable region that binds to a third antigen different from the first antigen and the second antigen.
[2] The heavy chain variable region can be bound to the first antigen and a second antigen different from the first antigen, but not simultaneously bound to the first antigen and the second antigen. An antigen-binding molecule comprising a variable region of an antibody in which amino acids have been modified.
[3] A variable region that does not bind to the first antigen and the second antigen simultaneously is a variable region that does not bind to the first antigen and the second antigen that are expressed on different cells, respectively. The antigen-binding molecule according to [1] or [2].
[4] The antigen-binding molecule according to any one of [1] to [3], further comprising an Fc region of the antibody.
[5] The antigen-binding molecule according to [4], wherein the Fc region has a reduced Fc region binding activity to FcγR compared to the Fc region of a natural human IgG1 antibody.
[6] The antigen-binding molecule according to any one of [1] to [5], which is a multispecific antibody.
[7] Any of [1] to [6], wherein the variable region of the antibody capable of binding to the first antigen and the second antigen is a variable region into which at least one amino acid modification has been introduced. The antigen-binding molecule described in 1.
[8] The antigen-binding molecule according to [7], wherein the modification is substitution or insertion of at least one amino acid.
[9] Substitution of the amino acid sequence of the variable region that binds to the first antigen to the amino acid sequence that binds to the second antigen, or the amino acid of the variable region that binds to the first antigen The antigen-binding molecule according to [7] or [8], which is an insertion of an amino acid sequence that binds to the second antigen into the sequence.
[10] The antigen-binding molecule according to [8] or [9], wherein the number of inserted amino acids is 1 to 25.
[11] The antigen-binding molecule according to any one of [7] to [10], wherein the amino acid to be modified is an amino acid of a CDR1, CDR2, CDR3, or FR3 region of an antibody variable region.
[12] The antigen-binding molecule according to any one of [7] to [11], wherein the amino acid to be modified is a loop region amino acid.
[13] The amino acids to be modified include Kabat numbering 31 to 35, 50 to 65, 71 to 74 and 95 to 102 of the heavy chain variable region of the antibody, and Kabat numbering 24 to 34, 50 to 56 and 89 of the light chain variable region. The antigen-binding molecule according to any one of [7] to [11], which is at least one amino acid selected from -97.
[14] Either one of the first antigen and the second antigen is a molecule that is specifically expressed on the surface of T cells, and the other antigen is expressed on the surface of T cells or other immune cells. The antigen-binding molecule according to any one of [1] to [13], which is a molecule.
[15] Either one of the first antigen or the second antigen is CD3, and the other antigen is FcγR, TLR, lectin, IgA, immune checkpoint molecule, TNF superfamily molecule, TNFR superfamily molecule or NK receptor The antigen-binding molecule according to [14], which is a molecule.
[16] The antigen-binding molecule according to [14] or [15], wherein the third antigen is a molecule expressed specifically in cancer tissue.
[17] A pharmaceutical composition comprising the antigen-binding molecule according to any one of [1] to [16] and a medically acceptable carrier.
[18] A method for producing the antigen-binding molecule according to any one of [1] to [16], comprising steps (i) to (iv):
(i) an antigen-binding molecule in which at least one amino acid of a variable region of an antibody that binds to the first antigen or the second antigen is modified, wherein at least one of the amino acids of the modified variable region is different from each other Creating a library of antigen binding molecules comprising the region,
(ii) an antigen comprising a variable region that has binding activity to the first antigen and the second antigen, but does not bind simultaneously with the first antigen and the second antigen, from the prepared library Selecting a binding molecule,
(iii) a host cell comprising a nucleic acid encoding the variable region of the antigen-binding molecule selected in step (ii) and / or a nucleic acid encoding the variable region of the antigen-binding molecule that binds to the third antigen. A variable region of an antibody that can bind to the first antigen and the second antigen, but does not bind to the first antigen and the second antigen at the same time, and / or the third antigen. Expressing an antigen binding molecule comprising a variable region that binds; and
(iv) recovering the antigen-binding molecule from the host cell culture.
[19] The first antigen and the second antigen that are contained in the antigen-binding molecule selected in step (ii) and that do not bind simultaneously to the first antigen and the second antigen are expressed on different cells. The production method according to [18], which is a variable region that does not bind to two antigens simultaneously.
[20] The production method according to [18] or [19], wherein the host cell cultured in step (iii) further comprises a nucleic acid encoding the Fc region of the antibody.
[21] The production method of [20], wherein the Fc region has a reduced Fc region binding activity to FcγR compared to the Fc region of a natural human IgG1 antibody.
[22] The production method according to any one of [18] to [21], wherein the antigen-binding molecule to be produced is a multispecific antibody.
[23] The production method according to any one of [18] to [22], wherein at least one modified amino acid in the variable region in step (i) is a substituted or inserted amino acid.
[24] The production method according to [23], wherein the number of inserted amino acids is 1 to 25.
[25] The production method according to any one of [18] to [24], wherein the modification is an amino acid modification of a CDR1, CDR2, CDR3, or FR3 region of an antibody variable region.
[26] The production method according to any one of [18] to [25], wherein the modification is modification of an amino acid in a loop region.
[27] Modifications from Kabat numbering 31-35, 50-65, 71-74 and 95-102 of the heavy chain variable region of the antibody, and Kabat numbering 24-34, 50-56 and 89-97 of the light chain variable region The production method according to any one of [18] to [25], which is a modification of at least one selected amino acid.
[28] Either one of the first antigen and the second antigen is a molecule that is specifically expressed on the surface of T cells, and the other antigen is expressed on the surface of T cells or other immune cells. [18] The production method according to any one of [18] to [27], which is a molecule.
[29] Either one of the first antigen and the second antigen is CD3, and the other antigen is FcγR, TLR, IgA, lectin, immune checkpoint molecule, TNF superfamily molecule, TNFR superfamily molecule or NK receptor [28] The production method according to [28], which is a molecule.
[30] The production method according to [28] or [29], wherein the third antigen is a molecule expressed specifically in cancer tissue.
[31] A method for treating cancer, comprising a step of administering the antigen-binding molecule according to any one of [1] to [16].
[32] The antigen-binding molecule according to any one of [1] to [16] for use in the treatment of cancer.
[33] Use of the antigen-binding molecule according to any one of [1] to [16] in the manufacture of a therapeutic agent for cancer.
[34] A process for producing a therapeutic agent for cancer, comprising a step of using the antigen-binding molecule according to any one of [1] to [16].
[35] Those skilled in the art will naturally understand that any combination of one or more of the above-described embodiments is also included in the present invention unless there is a technical contradiction based on the common general knowledge of those skilled in the art. The
ここで「異なる細胞上で発現」とは、別々の細胞上で発現していればよく、そのような細胞の組合せとしては、例えば、T細胞と別のT細胞のように同一種類の細胞であってもよいし、T細胞とNK細胞のように異なる種類の細胞であってもよい。 The “variable region of the antibody” of the present invention does not bind to the first antigen and the second antigen at the same time in the state where the variable region of the antibody of the present invention is bound to the first antigen. This means that it cannot bind to the second antigen, and conversely, when the variable region is bound to the second antigen, it cannot bind to the first antigen. Here, “does not bind to the first antigen and the second antigen at the same time” means that the two cells of the cell expressing the first antigen and the cell expressing the second antigen are cross-linked. Or not simultaneously binding to the first and second antigens expressed in separate cells. Furthermore, when the first antigen and the second antigen are not expressed on the cell membrane as in the soluble protein, or both are present on the same cell, the first antigen and the second antigen Although it is possible to bind to both antigens at the same time, cases where they cannot be simultaneously bound are also included when they are expressed on different cells. The variable region of such an antibody is not particularly limited as long as it has the function. For example, the variable region of a variable region of an IgG-type antibody may be modified so that it binds to a desired antigen. An area can be mentioned. As the amino acid to be modified, for example, an amino acid that does not lose binding to the antigen by amino acid modification is selected from the variable region of the antibody that binds to the first antigen or the second antigen.
Here, “expressed on different cells” is only required to be expressed on different cells. As a combination of such cells, for example, a T cell and another T cell can be the same type of cell. There may be different types of cells such as T cells and NK cells.
複数組み合わせて使用する場合、組み合わせる数は特に限定されず、発明の目的を達成できる範囲内で適宜設定することができ、例えば、2個以上30個以下、好ましくは2個以上25個以下、2個以上22個以下、2個以上20個以下、2個以上15個以下、2個以上10個以下、2個以上5個以下、2個以上3個以下である。
複数組み合わせる場合、抗体の重鎖可変領域又は軽鎖可変領域のみに当該アミノ酸改変を加えてもよく、重鎖可変領域と軽鎖可変領域の双方に適宜振り分けて加えてもよい。 The amino acid modification of the present invention may be used alone or in combination.
When used in combination, the number of combinations is not particularly limited, and can be set as appropriate within the range in which the object of the invention can be achieved. For example, 2 to 30 or less, preferably 2 to 25 or less, 2 2 or more and 20 or less, 2 or more and 15 or less, 2 or more and 10 or less, 2 or more and 5 or less, 2 or more and 3 or less.
In the case of combining a plurality, the amino acid modification may be added only to the heavy chain variable region or the light chain variable region of the antibody, or may be appropriately distributed to both the heavy chain variable region and the light chain variable region.
本発明においてアミノ酸の改変とは、置換、欠失、付加、挿入、あるいは修飾のいずれか、又はそれらの組み合わせを意味する。本発明においては、アミノ酸の改変はアミノ酸の変異と言い換えることが可能であり、同じ意味で使用される。 In the present invention, the “loop region” means a region where there are residues that are not involved in maintaining the β-barrel structure of immunoglobulin.
In the present invention, the amino acid modification means any one of substitution, deletion, addition, insertion, modification, or a combination thereof. In the present invention, amino acid modification can be rephrased as amino acid mutation and is used interchangeably.
アミノ酸残基は一般の側鎖の特性に基づいて以下のグループに分類される: (1) 疎水性: ノルロイシン、met、ala、val、leu、ile; (2) 中性親水性: cys、ser、thr、asn、gln; (3) 酸性: asp、glu; (4) 塩基性: his、lys、arg; (5) 鎖の配向に影響する残基: gly、pro;及び (6) 芳香族性: trp、tyr、phe。 When substituting an amino acid residue, the purpose is to modify, for example, the following points (a) to (c) by substituting with another amino acid residue. (a) the backbone structure of the polypeptide in the region of the sheet structure or helical structure; (b) the charge or hydrophobicity at the target site, or (c) the size of the side chain.
Amino acid residues are classified into the following groups based on general side chain properties: (1) Hydrophobicity: norleucine, met, ala, val, leu, ile; (2) Neutral hydrophilicity: cys, ser , Thr, asn, gln; (3) Acidity: asp, glu; (4) Basicity: his, lys, arg; (5) Residues that affect chain orientation: gly, pro; and (6) Aromatic Sex: trp, tyr, phe.
本発明における置換は、保存的置換であってもよく、非保存的置換であってもよく、また保存的置換と非保存的置換の組合せであってもよい。 Substitution of amino acid residues within each of these groups is called conservative substitution, while substitution of amino acid residues between other groups is called non-conservative substitution.
The substitution in the present invention may be a conservative substitution, a non-conservative substitution, or a combination of a conservative substitution and a non-conservative substitution.
例えば、電気化学発光法(ECL法)によって測定することができる(BMC Research Notes 2011, 4:281 )。
具体的には、例えば、ビオチン標識された被験抗原結合分子の第1の抗原と第2の抗原に結合することができる領域、例えば、Fab領域からなる低分子化された抗体、或いは、一価性の抗体(通常の抗体が有する2つのFab領域のうち1つがない抗体)に、sulfo-tag(Ru錯体)で標識された第1の抗原又は第2の抗原を混合し、ストレプトアビジン固相化プレート上に添加する。このとき、ビオチン標識された被検抗原結合分子はプレート上のストレプトアビジンに結合される。Sulfo-tagを発光させ、Sector Imager 600、2400(MSD)等を利用して、その発光シグナルを検出することで、第1の抗原又は第2の抗原と、被験抗原結合分子の上述の領域との結合を確認することができる。
また、ELISAやFACS(fluorescence activated cell sorting)、ALPHAスクリーン(Amplified Luminescent Proximity Homogeneous Assay)や表面プラズモン共鳴(SPR)現象を利用したBIACORE法等によって測定することもできる(Proc.Natl.Acad.Sci.USA (2006) 103 (11), 4005-4010)。 Whether or not the variable region of the antibody of the present invention can bind to the first antigen and the second antigen can be measured using a known method.
For example, it can be measured by an electrochemiluminescence method (ECL method) (BMC Research Notes 2011, 4: 281).
Specifically, for example, a region capable of binding to the first antigen and the second antigen of a test antigen-binding molecule labeled with biotin, for example, a low molecular weight antibody consisting of a Fab region, or monovalent 1st or 2nd antigen labeled with a sulfo-tag (Ru complex) is mixed with a natural antibody (an antibody that does not have one of the two Fab regions of a normal antibody), and a streptavidin solid phase Add onto the crystallization plate. At this time, the test antigen-binding molecule labeled with biotin is bound to streptavidin on the plate. By emitting Sulfo-tag and detecting the luminescence signal using
It can also be measured by ELISA, FACS (fluorescence activated cell sorting), ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), BIACORE method using surface plasmon resonance (SPR) phenomenon, etc. (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).
また、ELISAプレートあるいはセンサーチップに固定した第1の抗原又は第2の抗原のいずれか一方への抗原結合分子の結合が、他方を溶液中に添加することで阻害されるかどうかを測定することによって確認することもできる。 Whether or not the antigen-binding molecule of the present invention "does not bind to the first antigen and the second antigen at the same time" is confirmed after having confirmed the binding activity to the first antigen and the second antigen. Whether the antigen-binding molecule containing the variable region having the binding activity has binding activity for the remaining one after binding either the first antigen or the second antigen in advance. Can be confirmed by measuring using the method described above.
In addition, measuring whether the binding of the antigen-binding molecule to either the first antigen or the second antigen immobilized on the ELISA plate or the sensor chip is inhibited by adding the other to the solution. Can also be confirmed.
また、蛍光共鳴エネルギー移動(FRET; Fluorescence Resonance Energy Transfer)を利用した方法を用いることもできる。FRETとは、近接した2個の蛍光分子の間で励起エネルギーが、電子の共鳴により直接移動する現象である。FRETが起こると、ドナー (励起状態にある蛍光分子) の励起エネルギーがアクセプター (ドナーの近傍にあるもう1つの蛍光分子) に移動するため、ドナーから放射される蛍光が消失 (正確には蛍光寿命の短縮) し、代わりにアクセプターから蛍光が放射される。この現象を用いてdual-Fabであるかどうかを解析することができる。例えば、蛍光ドナーを導入した第1の抗原と、蛍光アクセプターを導入した第2の抗原が、被検抗原結合分子に同時に結合すると、ドナーの蛍光は消失し、アクセプターから蛍光が放射されるため、蛍光波長の変化が起こる。このような抗体はdual-Fabではないと判断される。一方、第1の抗原、第2の抗原、被検抗原結合分子を混合した際に、第1の抗原に結合させた蛍光ドナーの蛍光波長の変化が起こらなければ、この被検抗原結合分子はdual-Fabであるということができる。 In the case of the ALPHA screen, in the absence of a competing second antigen, the test antigen-binding molecule interacts with the first antigen to generate a signal of 520-620 nm. The untagged second antigen competes with the interaction between the test antigen binding molecule and the first antigen. Relative binding activity can be determined by quantifying the decrease in fluorescence that results from competition. It is known that a polypeptide is biotinylated using Sulfo-NHS-biotin or the like. As a method of tagging the first antigen with GST, the first antigen is expressed in a cell or the like holding a vector capable of expressing a fusion gene in which a polynucleotide encoding the first antigen and a polynucleotide encoding GST are fused in frame. In addition, a purification method using a glutathione column can be appropriately employed. The obtained signal is suitably analyzed by fitting to a one-site competition model using nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego). At this time, the same analysis can be performed by tagging the second antigen and not tagging the first antigen.
Further, a method using fluorescence resonance energy transfer (FRET) can be used. FRET is a phenomenon in which excitation energy moves directly between two adjacent fluorescent molecules by electron resonance. When FRET occurs, the excitation energy of the donor (excited fluorescent molecule) is transferred to the acceptor (another fluorescent molecule in the vicinity of the donor), and the fluorescence emitted from the donor disappears (exactly the fluorescence lifetime). Instead, fluorescence is emitted from the acceptor instead. It is possible to analyze whether this is a dual-Fab using this phenomenon. For example, when the first antigen introduced with a fluorescent donor and the second antigen introduced with a fluorescent acceptor are simultaneously bound to a test antigen-binding molecule, the donor's fluorescence disappears and the acceptor emits fluorescence. A change in fluorescence wavelength occurs. Such an antibody is judged not to be dual-Fab. On the other hand, when the first antigen, the second antigen, and the test antigen binding molecule are mixed, if the fluorescence wavelength of the fluorescent donor bound to the first antigen does not change, the test antigen binding molecule is It can be said that it is dual-Fab.
具体的には、第一の抗原と第二の抗原に対する同時結合を検出するECL-ELISAで陽性になった場合においても、それぞれ第一の抗原を発現する細胞と第二の抗原を発現する細胞及び被検抗原結合分子を混合した場合に、これら3者が同時に結合することがなければ、異なる細胞上に発現している場合には同時には結合することができないことを示すことができる。例えば、細胞を用いたECL-ELISA法で測定することが可能である。あらかじめ第一の抗原を発現する細胞をプレートに固相化し、被検抗原結合分子を結合させた後に、第二の抗原を発現する細胞を加える。第二の抗原を発現する細胞にのみ発現する別の抗原に対する、sulfo-tag標識抗体を用いて検出することにより、二つの細胞上に発現する二つの抗原と同時に結合している場合はシグナルが観測され、同時に結合しない場合は、シグナルが観測されない。
もしくは、alphascreen法で測定することが可能である。ドナービーズを結合した第1の抗原を発現する細胞と、アクセプタービーズを結合した第2の抗原を発現する細胞と、被検抗原結合分子を混合した際に、二つの細胞上に発現する二つの抗原と同時に結合している場合はシグナルが観測され、同時に結合しない場合は、シグナルが観測されない。
もしくはOctetを用いた相互作用解析法で測定することが可能である。まず初めに、ぺプチドタグを付加した第1の抗原を発現する細胞を、ペプチドタグを認識するバイオセンサーに結合させる。第二の抗原を発現する細胞と被検抗原結合分子を入れたウェルで相互作用解析を行った際に、二つの細胞上に発現する二つの抗原と同時に結合している場合は被検抗原結合分子と第2の抗原を発現する細胞がバイオセンサーに結合することにより大きな波長シフトが観測され、同時に結合しない場合は、被検抗原結合分子だけがバイオセンサーに結合するため、小さな波長シフトが観測される。 In addition, when the first antigen and the second antigen are not expressed on the cell membrane like a soluble protein, or when both are present on the same cell, the first antigen and the second antigen Can be simultaneously bound to both antigens, but when they are expressed on different cells, the case where they cannot be bound simultaneously can also be measured by using a known method.
Specifically, cells that express the first antigen and cells that express the second antigen, respectively, even when positive by ECL-ELISA that detects simultaneous binding to the first antigen and the second antigen. When the test antigen-binding molecules are mixed, if these three do not bind simultaneously, it can be shown that they cannot bind simultaneously when expressed on different cells. For example, it can be measured by an ECL-ELISA method using cells. Cells that express the first antigen are immobilized on a plate in advance and bound to the test antigen-binding molecule, and then cells that express the second antigen are added. By using a sulfo-tag-labeled antibody to detect another antigen that is expressed only in cells that express the second antigen, the signal is displayed when the two antigens expressed on the two cells are bound simultaneously. If observed and not bound simultaneously, no signal is observed.
Alternatively, it can be measured by the alphascreen method. When a test antigen-binding molecule is mixed with a cell expressing a first antigen bound to a donor bead, a cell expressing a second antigen bound to an acceptor bead, A signal is observed when bound simultaneously with two antigens, and no signal is observed when bound simultaneously.
Alternatively, it can be measured by an interaction analysis method using Octet. First, cells expressing a first antigen to which a peptide tag has been added are bound to a biosensor that recognizes a peptide tag. When an interaction analysis is performed in a well containing a second antigen-expressing cell and a test antigen-binding molecule, if the two antigens expressed on two cells are bound simultaneously, the test antigen binding A large wavelength shift is observed when the cell expressing the molecule and the second antigen binds to the biosensor. If the cells do not bind simultaneously, only the test antigen-binding molecule binds to the biosensor, so a small wavelength shift is observed. Is done.
FcγRIのポリヌクレオチド配列及びアミノ酸配列は、それぞれNM_000566.3及びNP_000557.1に、FcγRIIaのポリヌクレオチド配列及びアミノ酸配列は、それぞれBC020823.1及びAAH20823.1に、FcγRIIbのポリヌクレオチド配列及びアミノ酸配列は、それぞれBC146678.1及びAAI46679.1に、FcγRIIIaのポリヌクレオチド配列及びアミノ酸配列は、それぞれBC033678.1及びAAH33678.1に、並びにFcγRIIIbのポリヌクレオチド配列及びアミノ酸配列は、それぞれBC128562.1及びAAI28563.1に記載されている(RefSeq登録番号)。尚、FcγRIIaには、FcγRIIaの131番目のアミノ酸がヒスチジン(H型)あるいはアルギニン(R型)に置換された2種類の遺伝子多型が存在する(J. Exp. Med, 172, 19-25, 1990)。また、FcγRIIbには、FcγRIIbの232番目のアミノ酸がイソロイシン(I型)あるいはスレオニン (T型)に置換された2種類の遺伝子多型が存在する(Arthritis. Rheum. 46: 1242-1254 (2002))。また、FcγRIIIaには、FcγRIIIaの158番目のアミノ酸がバリン(V型)あるいはフェニルアラニン(F型)に置換された2種類の遺伝子多型が存在する(J. Clin. Invest. 100(5): 1059-1070 (1997))。また、FcγRIIIbには、NA1型、NA2型の2種類の遺伝子多型が存在する(J. Clin. Invest. 85: 1287-1295 (1990))。 FcγR has an active receptor having an ITAM (immunoreceptor tyrosine-based activation motif) and an inhibitory receptor having an ITIM (immunoreceptor tyrosine-based inhibitory motif). FcγR is classified into FcγRI, FcγRIIa R, FcγRIIa H, FcγRIIIa, and FcγRIIIb active FcγR, and FcγRIIb inhibitory FcγR.
The polynucleotide sequence and amino acid sequence of FcγRI are NM_000566.3 and NP_000557.1, respectively.The polynucleotide sequence and amino acid sequence of FcγRIIa are BC020823.1 and AAH20823.1, respectively.The polynucleotide sequence and amino acid sequence of FcγRIIb are BC146678.1 and AAI46679.1, respectively, FcγRIIIa polynucleotide sequence and amino acid sequence are BC033678.1 and AAH33678.1, respectively, and FcγRIIIb polynucleotide sequence and amino acid sequence are BC128562.1 and AAI28563.1, respectively. It is listed (RefSeq registration number). FcγRIIa has two gene polymorphisms in which the 131st amino acid of FcγRIIa is substituted with histidine (H type) or arginine (R type) (J. Exp. Med, 172, 19-25, 1990). In addition, FcγRIIb has two gene polymorphisms in which the 232nd amino acid of FcγRIIb is replaced with isoleucine (type I) or threonine (type T) (Arthritis. Rheum. 46: 1242-1254 (2002) ). In addition, FcγRIIIa has two gene polymorphisms in which the 158th amino acid of FcγRIIIa is substituted with valine (V type) or phenylalanine (F type) (J. Clin. Invest. 100 (5): 1059 -1070 (1997)). FcγRIIIb has two gene polymorphisms, NA1 type and NA2 type (J. Clin. Invest. 85: 1287-1295 (1990)).
ALPHAスクリーンは、ドナーとアクセプターの2つのビーズを使用するALPHAテクノロジーによって下記の原理に基づいて実施される。ドナービーズに結合した分子が、アクセプタービーズに結合した分子と生物学的に相互作用し、2つのビーズが近接した状態の時にのみ、発光シグナルを検出される。レーザーによって励起されたドナービーズ内のフォトセンシタイザーは、周辺の酸素を励起状態の一重項酸素に変換する。一重項酸素はドナービーズ周辺に拡散し、近接しているアクセプタービーズに到達するとビーズ内の化学発光反応を引き起こし、最終的に光が放出される。ドナービーズに結合した分子とアクセプタービーズに結合した分子が相互作用しないときは、ドナービーズの産生する一重項酸素がアクセプタービーズに到達しないため、化学発光反応は起きない。 Whether Fcγ receptor binding activity is reduced should be confirmed by well-known methods such as FACS, ELISA format, ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) and BIACORE method using surface plasmon resonance (SPR) phenomenon. (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).
The ALPHA screen is implemented according to the following principle by ALPHA technology using two beads of donor and acceptor. A molecule bound to the donor bead interacts biologically with the molecule bound to the acceptor bead, and a luminescent signal is detected only when the two beads are in close proximity. A photosensitizer in the donor bead excited by the laser converts ambient oxygen into excited singlet oxygen. Singlet oxygen diffuses around the donor bead, and when it reaches the adjacent acceptor bead, it causes a chemiluminescence reaction in the bead, and finally light is emitted. When the molecule bound to the donor bead and the molecule bound to the acceptor bead do not interact, the chemiluminescence reaction does not occur because the singlet oxygen produced by the donor bead does not reach the acceptor bead.
対照とする抗原結合分子としては、IgG1、IgG2、IgG3又はIgG4モノクローナル抗体のFc領域を有する抗原結合分子が適宜使用され得る。当該Fc領域の構造は、配列番号:1(RefSeq登録番号AAC82527.1のN末にA付加)、配列番号:2(RefSeq登録番号AAB59393.1のN末にA付加)、配列番号:3(RefSeq登録番号CAA27268.1のN末にA付加)、配列番号:4(RefSeq登録番号AAB59394.1のN末にA付加)に記載されている。また、ある特定のアイソタイプの抗体のFc領域の変異体を有する抗原結合分子を被検物質として使用する場合には、当該特定のアイソタイプの抗体のFc領域を有する抗原結合分子を対照として用いることによって、当該変異体が有する変異によるFcγ受容体への結合活性に対する効果が検証される。上記のようにして、Fcγ受容体に対する結合活性が低下していることが検証されたFc領域の変異体を有する抗原結合分子が適宜作製される。 In the present specification, the fact that the binding activity to the Fcγ receptor is reduced is, for example, based on the above analysis method, compared to the binding activity of the antigen-binding molecule containing the Fc region as a control, compared to the test antigen binding Binding activity of molecules is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, 15% or less, particularly preferably 10% or less, 9% or less, 8% or less 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.
As a control antigen-binding molecule, an antigen-binding molecule having an Fc region of IgG1, IgG2, IgG3 or IgG4 monoclonal antibody can be used as appropriate. The structure of the Fc region is as follows. RefSeq registration number CAA27268.1 with an A added at the N terminus), SEQ ID NO: 4 (RefSeq registration number AAB59394.1 with an N added at the N ending). In addition, when an antigen-binding molecule having a variant of the Fc region of an antibody of a specific isotype is used as a test substance, the antigen-binding molecule having the Fc region of the antibody of the specific isotype is used as a control. The effect of the mutation of the mutant on the binding activity to the Fcγ receptor is verified. As described above, an antigen-binding molecule having a variant of the Fc region that has been verified to have reduced binding activity to the Fcγ receptor is appropriately prepared.
すなわち、特定のアイソタイプの抗体のFc領域を構成するアミノ酸のうち、EUナンバリングに従って特定される下記のいずれかのアミノ酸;220位、226位、229位、231位、232位、233位、234位、235位、236位、237位、238位、239位、240位、264位、265位、266位、267位、269位、270位、295位、296位、297位、298位、299位、300位、325位、327位、328位、329位、330位、331位、332位が置換されているFc領域を有する抗原結合分子が好適に挙げられる。Fc領域の起源である抗体のアイソタイプとしては特に限定されず、IgG1、IgG2、IgG3又はIgG4モノクローナル抗体を起源とするFc領域が適宜利用され得るが、天然型ヒトIgG1抗体を起源とするFc領域が好適に利用される。
例えば、IgG1抗体のFc領域を構成するアミノ酸のうち、EUナンバリングに従って特定される下記のいずれかの置換(数字がEUナンバリングに従って特定されるアミノ酸残基の位置、数字の前に位置する一文字のアミノ酸記号が置換前のアミノ酸残基、数字の後に位置する一文字のアミノ酸記号が置換前のアミノ酸残基をそれぞれ表す);
(a)L234F、L235E、P331S、
(b)C226S、C229S、P238S、
(c)C226S、C229S、
(d)C226S、C229S、E233P、L234V、L235A
が施されているFc領域、又は、231位から238位のアミノ酸配列が欠失したFc領域を有する抗原結合分子も適宜使用され得る。 Examples of such variants include deletion of amino acids 231A-238S identified according to EU numbering (WO 2009/011941), C226S, C229S, P238S, (C220S) (J. Rheumatol (2007) 34, 11), C226S, C229S (Hum.Antibod.Hybridomas (1990) 1 (1), 47-54), C226S, C229S, E233P, L234V, L235A (Blood (2007) 109, 1185-1192) It is known.
That is, among the amino acids constituting the Fc region of an antibody of a specific isotype, any one of the following amino acids specified according to EU numbering: positions 220, 226, 229, 231, 232, 233, 234 , 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 297, 298, 299 Preferred examples include antigen-binding molecules having Fc regions in which the position,
For example, among the amino acids constituting the Fc region of IgG1 antibody, one of the following substitutions specified according to EU numbering (position of amino acid residue specified according to EU numbering, one-letter amino acid positioned before the number) The symbol is the amino acid residue before substitution, and the one-letter amino acid symbol located after the number represents the amino acid residue before substitution);
(A) L234F, L235E, P331S,
(B) C226S, C229S, P238S,
(C) C226S, C229S,
(D) C226S, C229S, E233P, L234V, L235A
Or an antigen-binding molecule having an Fc region in which the amino acid sequence from positions 231 to 238 has been deleted can be used as appropriate.
(e)H268Q、V309L、A330S、P331S
(f)V234A
(g)G237A
(h)V234A、G237A
(i)A235E、G237A
(j)V234A、A235E、G237A
が施されているFc領域を有する抗原結合分子も適宜使用され得る。 In addition, among the amino acids constituting the Fc region of IgG2 antibody, one of the following substitutions specified according to EU numbering (position of amino acid residue specified according to EU numbering, one-letter amino acid positioned before the number) The symbol is the amino acid residue before substitution, and the one-letter amino acid symbol located after the number represents the amino acid residue before substitution);
(E) H268Q, V309L, A330S, P331S
(F) V234A
(G) G237A
(H) V234A, G237A
(I) A235E, G237A
(J) V234A, A235E, G237A
Antigen-binding molecules having an Fc region that has been subjected to can be used as appropriate.
(k)F241A
(l)D265A
(m)V264A
が施されているFc領域を有する抗原結合分子も適宜使用され得る。 In addition, among the amino acids constituting the Fc region of IgG3 antibody, one of the following substitutions specified according to EU numbering (number of amino acid residue specified according to EU numbering, one-letter amino acid located before the number) The symbol is the amino acid residue before substitution, and the one-letter amino acid symbol located after the number represents the amino acid residue before substitution);
(K) F241A
(L) D265A
(M) V264A
Antigen-binding molecules having an Fc region that has been subjected to can be used as appropriate.
(n)L235A、G237A、E318A
(o)L235E
(p)F234A、L235A
が施されているFc領域を有する抗原結合分子も適宜使用され得る。 In addition, among the amino acids constituting the Fc region of IgG4 antibody, one of the following substitutions specified according to EU numbering (position of amino acid residue specified according to EU numbering, one-letter amino acid located before the number) The symbol is the amino acid residue before substitution, and the one-letter amino acid symbol located after the number represents the amino acid residue before substitution);
(N) L235A, G237A, E318A
(O) L235E
(P) F234A, L235A
Antigen-binding molecules having an Fc region that has been subjected to can be used as appropriate.
CH2又はCH3の界面に電荷的な反発を導入して意図しないH鎖同士の会合を抑制させる技術において、H鎖の他の定常領域の界面で接触するアミノ酸残基としては、例えばCH3領域におけるEUナンバリング356番目の残基、EUナンバリング439番目の残基、EUナンバリング357番目の残基、EUナンバリング370番目の残基、EUナンバリング399番目の残基、EUナンバリング409番目の残基に相対する領域を挙げることができる。 For the purpose of associating multispecific antibodies, undesired heavy chains are introduced by introducing a charge repulsion at the interface of the second constant region (CH2) of the antibody heavy chain or the third constant region (CH3) of the heavy chain. A technique for suppressing the association between each other can be applied (WO2006 / 106905).
In the technique of suppressing unintentional association of H chains by introducing charge repulsion to the CH2 or CH3 interface, amino acid residues that contact at the interface of other constant regions of the H chain include, for example, EU in the CH3 region Numbering region 356, EU numbering 439th residue, EU numbering 357th residue, EU numbering 370th residue, EU numbering 399th residue, EU numbering 409th residue Can be mentioned.
(a)グルタミン酸(E)、アスパラギン酸(D)、
(b)リジン(K)、アルギニン(R)、ヒスチジン(H)。 In the above antibody, the “charged amino acid residue” is preferably selected from, for example, amino acid residues included in any of the following groups (a) or (b);
(A) glutamic acid (E), aspartic acid (D),
(B) Lysine (K), arginine (R), histidine (H).
本発明において改変に供するアミノ酸残基としては、上述した抗体の可変領域または抗体の定常領域のアミノ酸残基に限られない。当業者であれば、ポリペプチド変異体または異種多量体について、市販のソフトウェアを用いたホモロジーモデリング等により、界面を形成するアミノ酸残基を見出すことができ、会合を制御するように、該部位のアミノ酸残基を改変に供することが可能である。 In a preferred embodiment, in the antibody, the first H chain CH3 region and the second H chain CH3 region may be cross-linked by a disulfide bond.
The amino acid residues to be modified in the present invention are not limited to the above-described antibody variable region or antibody constant region amino acid residues. A person skilled in the art can find amino acid residues that form an interface for polypeptide variants or heterologous multimers by homology modeling using commercially available software, etc. Amino acid residues can be subjected to modification.
同一ポリペプチド鎖上にコードされるVLとVHとは、その間のリンカーが短いため単鎖可変領域フラグメントを形成することが出来ず、二量体化することにより、2つの抗原結合部位を形成する。 Db is a dimer composed of two polypeptide chains (Holliger P et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), EP404, 097, W093 / 11161, etc.) In each polypeptide chain, the L chain variable region (VL) and the H chain variable region (VH) in the same chain are connected to each other by a short linker, for example, about 5 residues. Yes.
VL and VH encoded on the same polypeptide chain cannot form a single-chain variable region fragment due to the short linker between them, and form two antigen-binding sites by dimerization. .
[VL]リンカー[VH]リンカー[VH]リンカー[VL]
[VH]リンカー[VL]リンカー[VL]リンカー[VH]
[VH]リンカー[VH]リンカー[VL]リンカー[VL]
[VL]リンカー[VL]リンカー[VH]リンカー[VH]
[VL]リンカー[VH]リンカー[VL]リンカー[VH] In the present specification, the antigen-binding domain constituting sc (Fv) 2 is composed of two VHs and two VLs, VH, VL, VH, VL ([[ VH] Linker [VL] Linker [VH] Linker [VL]) are listed in this order, but the order of two VHs and two VLs is not particularly limited to the above configuration, They may be arranged in any order. For example, the following configuration can be given.
[VL] Linker [VH] Linker [VH] Linker [VL]
[VH] Linker [VL] Linker [VL] Linker [VH]
[VH] Linker [VH] Linker [VL] Linker [VL]
[VL] Linker [VL] Linker [VH] Linker [VH]
[VL] Linker [VH] Linker [VL] Linker [VH]
Ser
Gly・Ser
Gly・Gly・Ser
Ser・Gly・Gly
Gly・Gly・Gly・Ser(配列番号:5)
Ser・Gly・Gly・Gly(配列番号:6)
Gly・Gly・Gly・Gly・Ser(配列番号:7)
Ser・Gly・Gly・Gly・Gly(配列番号:8)
Gly・Gly・Gly・Gly・Gly・Ser(配列番号:9)
Ser・Gly・Gly・Gly・Gly・Gly(配列番号:10)
Gly・Gly・Gly・Gly・Gly・Gly・Ser(配列番号:11)
Ser・Gly・Gly・Gly・Gly・Gly・Gly(配列番号:12)
(Gly・Gly・Gly・Gly・Ser(配列番号:7))n
(Ser・Gly・Gly・Gly・Gly(配列番号:8))n
[nは1以上の整数である]等を挙げることができる。但し、ペプチドリンカーの長さや配列は目的に応じて当業者が適宜選択することができる。 For example, for a peptide linker:
Ser
Gly ・ Ser
Gly ・ Gly ・ Ser
Ser ・ Gly ・ Gly
Gly, Gly, Gly, Ser (SEQ ID NO: 5)
Ser, Gly, Gly, Gly (SEQ ID NO: 6)
Gly, Gly, Gly, Gly, Ser (SEQ ID NO: 7)
Ser, Gly, Gly, Gly, Gly (SEQ ID NO: 8)
Gly, Gly, Gly, Gly, Gly, Ser (SEQ ID NO: 9)
Ser, Gly, Gly, Gly, Gly, Gly (SEQ ID NO: 10)
Gly, Gly, Gly, Gly, Gly, Gly, Ser (SEQ ID NO: 11)
Ser, Gly, Gly, Gly, Gly, Gly, Gly (SEQ ID NO: 12)
(Gly, Gly, Gly, Gly, Ser (SEQ ID NO: 7)) n
(Ser, Gly, Gly, Gly, Gly (SEQ ID NO: 8)) n
[N is an integer of 1 or more]. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art according to the purpose.
4つの抗体可変領域を結合する場合には、通常、3つのリンカーが必要となるが、全て同じリンカーを用いてもよいし、異なるリンカーを用いてもよい。 Synthetic chemical linkers (chemical crosslinkers) are commonly used to crosslink peptides such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl) Suberate (BS3), dithiobis (succinimidyl propionate) (DSP), dithiobis (sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis (succinimidyl succinate) (EGS), ethylene Glycol bis (sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis [2- (succinimideoxycarbonyloxy ) Ethyl] sulfone (BSOCOES), bis [2- (sulfosuccinimidooxycarbonyloxy) ethyl And the like sulfone (sulfo-BSOCOES), These crosslinking agents are commercially available.
When four antibody variable regions are linked, usually three linkers are required, but all may use the same linker or different linkers.
例えば、本発明の抗原結合分子には、さらに当該分子の目的とする機能に実質的な変化を与えない範囲で、任意に改変を加えることができる。例えばこのような変異はアミノ酸残基の保存的置換によって行うことができる。また、本発明の抗原結合分子の目的とする機能に変化を与えるような改変であっても、当該機能の変化が本発明の目的の範囲内であれば、そのような改変も行うことができる。 Further, the antigen-binding molecule of the present invention can further contain additional modifications in addition to the amino acid modifications described above. Additional alterations can be selected from, for example, any of amino acid substitutions, deletions, modifications, or combinations thereof.
For example, the antigen-binding molecule of the present invention can be arbitrarily modified as long as it does not substantially change the target function of the molecule. For example, such mutations can be made by conservative substitution of amino acid residues. Moreover, even if the modification gives a change to the target function of the antigen-binding molecule of the present invention, such a modification can be made as long as the change in the function is within the scope of the present invention. .
・哺乳動物の抗体産生細胞
・抗体をコードするDNAを含む発現ベクターで形質転換された真核細胞
ここに示した真核細胞には、酵母や動物細胞が含まれる。たとえばCHO細胞やHEK293H細胞は、抗体をコードするDNAを含む発現ベクターで形質転換するための代表的な動物細胞である。他方、当該位置に糖鎖修飾が無いものも本発明の抗体に含まれる。定常領域が糖鎖で修飾されていない抗体は、抗体をコードする遺伝子を大腸菌などの原核細胞で発現させて得ることができる。 The modification of the amino acid sequence in the present invention includes post-translational modification. As specific post-translational modifications, addition or deletion of sugar chains can be shown. For example, when the antigen-binding molecule of the present invention has an IgG1-type constant region, the 297th amino acid residue of EU numbering can be modified with a sugar chain. The sugar chain structure to be modified is not limited. In general, antibodies expressed in eukaryotic cells contain glycosylation in the constant region. Therefore, antibodies expressed in the following cells are usually modified with some sugar chain.
・ Mammalian antibody-producing cells
-Eukaryotic cells transformed with an expression vector containing DNA encoding the antibody. The eukaryotic cells shown here include yeast and animal cells. For example, CHO cells and HEK293H cells are representative animal cells for transformation with an expression vector containing DNA encoding an antibody. On the other hand, those having no sugar chain modification at the position are also included in the antibody of the present invention. An antibody whose constant region is not modified with a sugar chain can be obtained by expressing a gene encoding the antibody in a prokaryotic cell such as Escherichia coli.
asminogen activator inhibitor-1, platelet-growth factor, plgR, PLP, poly glycol chains of different size(e.g. PEG-20, PEG-30, PEG40), PP14, prekallikrein, prion protein, procalcitonin, Programmed cell death protein 1, proinsulin, prolactin, Proprotein convertase PC9, prorelaxin, prostate specific membrane antigen (PSMA), Protein A, Protein C, Protein D, Protein S, Protein Z, PS, PSA, PSCA, PsmAr, PTEN, PTHrp, Ptk, PTN, P-selectin glycoprotein ligand-1, R51, RAGE, RANK, RANKL, RANTES, relaxin, Relaxin A-chain, Relaxin B-chain, renin, respiratory syncytial virus (RSV) F, Ret, reticulon 4, Rheumatoid factors, RLI P76, RPA2, RPK-1, RSK, RSV Fgp, S100, RON-8, SCF/KL, SCGF, Sclerostin, SDF-1, SDF1α, SDF1β, SERINE, Serum Amyloid P, Serum albumin, sFRP-3, Shh, Shiga like toxin II, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, sphingosine 1-phosphate receptor 1, Staphylococcal lipoteichoic acid, Stat, STEAP, STEAP-II, stem cell factor (SCF), streptokinase, superoxide dismutase, syndecan-1, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TB, TCA-3, T-cell receptor alpha/beta, TdT, TECK, TEM1, TEM5, TEM7, TEM8, Tenascin, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta Rl (ALK-5), TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, TGF-I, Thrombin, thrombopoietin (TPO), Thymic stromal lymphoprotein receptor, Thymus Ck-1, thyroid stimulating hormone (TSH), thyroxine, thyroxine-binding globulin, Tie, TIMP, TIQ, Tissue Factor, tissue factor protease inhibitor, tissue factor protein, TMEFF2, Tmpo, TMPRSS2, TNF receptor I, TNF receptor II, TNF-alpha, TNF-beta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2/DR4), TNFRSF10B (TRAIL R2 DR5/KILLER/TRICK-2A/TRICK-B), TNFRSF10C (TRAIL R3 DcR1/LIT/TRID), TNFRSF10D (TRAIL R4 DcR2/TRUNDD), TNFRSF11A (RANK ODF R/TRANCE R), TNFRSF11B (OPG OCIF/TR1), TNFRSF12 (TWEAK R FN14), TNFRSF12A, TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR/HveA/LIGHT R/TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ/TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF Rl CD120a/p55-60), TNFRSF1B (TNF RII CD120b/p75-80), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRSF25 (DR3 Apo-3/LARD/TR-3/TRAMP/WSL-1), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII/TNFC R), TNFRSF4 (OX40 ACT35/TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1/APT1/CD95), TNFRSF6B (DcR3 M68/TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1 BB CD137/ILA), TNFRST23 (DcTRAIL R1 TNFRH1), TNFSF10 (TRAIL Apo-2 Ligand/TL2), TNFSF11 (TRANCE/RANK Ligand ODF/OPG Ligand), TNFSF12 (TWEAK Apo-3 Ligand/DR3 Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS/TALL1/THANK/TNFSF20), TNFSF14 (LIGHT HVEM Ligand/LTg), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR Ligand AITR Ligand/TL6), TNFSF1A (TNF-a Conectin/DIF/TNFSF2), TNFSF1B (TNF-b LTa/TNFSF1), TNFSF3 (LTb TNFC/p33), TNFSF4 (OX40 Ligand gp34/TXGP1), TNFSF5 (CD40 Ligand CD154/gp39/HIGM1/IMD3/TRAP), TNFSF6 (Fas Ligand Apo-1 Ligand/APT1 Ligand), TNFSF7 (CD27 Ligand CD70), TNFSF8 (CD30 Ligand CD153), TNFSF9 (4-1 BB Ligand CD137 Ligand), TNF-α, TNF-β, TNIL-I, toxic metabolite, TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, transforming growth factors (TGF) such as TGF-alpha and TGF-beta, Transmembrane glycoprotein NMB, Transthyretin, TRF, Trk, TROP-2, Trophoblast glycoprotein, TSG, TSLP, Tumor Necrosis Factor (TNF), tumor-associated antigen CA 125, tumor-associated antigen expressing Lewis Y related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VAP-1, vascular endothelial growth factor (VEGF), vaspin, VCAM, VCAM-1, VECAD, VE-Cadherin, VE-Cadherin-2, VEFGR-1 (flt-1), VEFGR-2, VEGF receptor (VEGFR), VEGFR-3 (flt-4), VEGI, VIM, Viral antigens, VitB12 receptor, Vitronectin receptor, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand Factor (vWF), WIF-1, WNT1, WNT10A, WNT10B, WNT11, WNT16, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1, XCL2/SCM-l-beta, XCLl/Lymphotactin, XCR1, XEDAR, XIAP, XPDなどがあげられる。 In the present specification, the “antigen” is not particularly limited and may be any antigen. Examples of antigens include 17-IA, 4-1 BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 Adenosine Receptor, A33, ACE, ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C, Activin RIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Addressins, adiponectin, ADP ribosyl cyclase-1, aFGF, AGE, ALCAM, ALK, ALK-1, ALK-7, allergen, alpha1-antichemotrypsin, alpha1-antitrypsin, alpha-synuclein, alpha-V / beta- 1 antagonist, aminin, amylin, amyloid beta, amyloid immunoglobulin heavy chain variable region.amyloid immunoglobulin light chain variable region, Androgen, ANG, angiotensinogen, Angiopoietin ligand-2, anti-Id, antithrombinIII, Anthrax, APAF-1, APE, APJ , apo A1, apo serum amyloid A, Apo-SAA, APP, APRIL, AR, ARC, ART, Artemin, ASPARTIC, Atrial natriuretic factor, Atrial natriuretic peptide, atrial natriuretic peptides A, atrial natriuretic peptides B, atrial natriuretic peptides C, av / b3 integrin, Axl, B7-1, B7-2, B7-H, BACE, BACE-1, Bacillus anthracis protective antigen, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, BcI, BCMA, BDNF, b-ECGF, beta-2-microglobulin, betalactamase, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, B-lymphocyte Stimulator (BIyS), BMP, BMP-2 (BMP- 2a), BMP-3 (Osteogenin), BMP-4 (BMP-2b), BMP-5, BMP-6 (Vgr-1), BMP-7 (OP-1), BMP-8 (BMP-8a), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BMPR-II (BRK-3), BMPs, BOK, Bombesin, Bone-derived neurotrophic factor, bovine growth hormone, BPDE, BPDE-DNA , BRK-2, BTC, B-lymphocyte cell adhesion molecule, C10, C1-inhibitor, C1q, C3, C3a, C4, C5, C5a (complement 5a), CA125, CAD-8, Cadherin-3, Calcitonin, cAMP, Carbonic anhydrase-IX, carcinoembryonic antigen (CEA), carcinoma-associated antigen, Cardiotrophin-1, Cathepsin A, Cathepsin B, Cathepsin C / DPPI, Cathepsin D, Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S, Cathepsin V, Cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1 / I-309, CCL11 / Eotaxin, CCL12 / MCP-5, CCL13 / MCP-4, CCL14 / HCC-1, CCL15 / HCC-2, CCL16 / HCC-4, CCL17 / TARC, CCL18 / PARC, CCL19 / ELC, CCL2 / MCP-1, CCL20 / MIP-3-alpha, CCL21 / SLC, CCL22 / MDC, CCL23 / MPIF-1, CCL24 / Eotaxin-2, CCL25 / TECK, CCL26 / Eotaxin-3, CCL27 / CTACK, CCL28 / MEC, CCL3 / M1P-1-alpha, CCL3Ll / LD- 78-beta, CCL4 / MIP-l-beta, CCL5 / RANTES, CCL6 / C10, CCL7 / MCP-3, CCL8 / MCP-2, CCL9 / 10 / MTP-1-gamma, CCR, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD10, CD105, CD11a, CD11b, CD11c, CD123, CD13, CD137, CD138, CD14, CD140a, CD146, CD147, CD148, CD15, CD152, CD16, CD164, CD18, CD19, CD2, CD20, CD21, CD22, CD23, CD25, CD26, CD27L, CD28, CD29, CD3, CD30, CD30L, CD32, CD33 (p67 proteins), CD34, CD37, CD38, CD3E, CD4, CD40, CD40L, CD44, CD45, CD46, CD49a, CD49b, CD5, CD51, CD52, CD54, CD55, CD56, CD6, CD61, CD64, CD66e, CD7, CD70, CD74, CD8, CD80 (B7-1), CD89 , CD95, CD105, CD158a, CEA, CEACAM5, CFTR, cGMP, CGRP receptor, CINC, CKb8-1, Claudin18, CLC, Clostridium botulinum toxin, Clostridium difficile toxin, Clostridium perfring ens toxin, c-Met, CMV, CMV UL, CNTF, CNTN-1, complement factor 3 (C3), complement factor D, corticosteroid-binding globulin, Colony stimulating factor-1 receptor, COX, C-Ret, CRG-2 , CRTH2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1 / Fractalkine, CX3CR1, CXCL, CXCL1 / Gro-alpha, CXCL10, CXCL11 / I-TAC, CXCL12 / SDF-l-alpha / beta, CXCL13 / BCA -1, CXCL14 / BRAK, CXCL15 / Lungkine. CXCL16, CXCL16, CXCL2 / Gro-beta CXCL3 / Gro-gamma, CXCL3, CXCL4 / PF4, CXCL5 / ENA-78, CXCL6 / GCP-2, CXCL7 / NAP-2, CXCL8 / IL-8, CXCL9 / Mig, CXCLlO / IP-10, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cystatin C, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decay accelerating factor, Delta-like protein ligand 4, des (1-3) -IGF-1 (brain IGF-1), Dhh, DHICA oxidase, Dickkopf-1, digoxin, Dipeptidyl peptidase IV, DKl, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EGF like domain containing protein 7, Elastase, elastin, EMA, EMMPRIN, ENA, ENA- 78, Endosialin, endothelin receptor, endotoxin, E nkephalinase, eNOS, Eot, Eotaxin, Eotaxin-2, eotaxini, EpCAM, Ephrin B2 / EphB4, Epha2 tyrosine kinase receptor, epidermal growth factor receptor (EGFR), ErbB2 receptor, ErbB3 tyrosine kinase receptor, ERCC, EREG, erythropoietin (EPO) , Erythropoietin receptor, E-selectin, ET-1, Exodus-2, F protein of RSV, F10, F11, F12, F13, F5, F9, Factor Ia, Factor IX, Factor Xa, Factor VII, factor VIII, Factor VIIIc , Fas, FcalphaR, FcepsilonRI, FcgammaIIb, FcgammaRI, FcgammaRIIa, FcgammaRIIIa, FcgammaRIIIb, FcRn, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF-2 receptor, FGF-3, FGF-8, FGF- acidic, FGF-basic, FGFR, FGFR-3, Fibrin, fibroblast activation protein (FAP), fibroblast growth factor, fibroblast growth factor-10, fibronectin, FL, FLIP, Flt-3, FLT3 ligand, Folate receptor, follicle stimulating hormone (FSH), Fractalkine (CX3C), free heavy chain, free light chain, FZD1, FZD10, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, G250, Gas 6, GCP-2, GCSF, G- CSF, G-CSF receptor, GD2, GD3, GDF, GDF-1, GDF-15 (M IC-1), GDF-3 (Vgr-2), GDF-5 (BMP-14 / CDMP-1), GDF-6 (BMP-13 / CDMP-2), GDF-7 (BMP-12 / CDMP- 3), GDF-8 (Myostatin), GDF-9, GDNF, Gelsolin, GFAP, GF-CSF, GFR-alpha1, GFR-alpha2, GFR-alpha3, GF-β1, gH envelope glycoprotein, GITR, Glucagon, Glucagon receptor , Glucagon-like peptide 1 receptor, Glut 4, Glutamate carboxypeptidase II, glycoprotein hormone receptors, glycoprotein IIb / IIIa (GP IIb / IIIa), Glypican-3, GM-CSF, GM-CSF receptor, gp130, gp140, gp72, granulocyte -CSF (G-CSF), GRO / MGSA, Growth hormone releasing factor, GRO-β, GRO-γ, H. pylori, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCC 1, HCMV gB envelope glycoprotein, HCMV UL, Hemopoietic growth factor (HGF), Hep B gp120, heparanase, heparin cofactor II, hepatic growth factor, Bacillus anthracis protective antigen, Hepatitis C virus E2 glycoprotein, Hepatitis E, Hepcidin, Her1, Her2 / neu ( ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HGF, HGFA, High molecular weight melanoma-associated antigen (HMW-MA A), HIV envelope proteins such as GP120, HIV MIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HMGB-1, HRG, Hrk, HSP47, Hsp90, HSV gD glycoprotein, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (hGH), human serum albumin, human tissue-type plasminogen activator (t-PA), Huntingtin, HVEM, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFN -alpha, IFN-beta, IFN-gamma, IgA, IgA receptor, IgE, IGF, IGF binding proteins, IGF-1, IGF-1 R, IGF-2, IGFBP, IGFR, IL, IL-1, IL-10 , IL-10 receptors, IL-11, IL-11 receptors, IL-12, IL-12 receptors, IL-13, IL-13 receptors, IL-15, IL-15 receptors, IL-16, IL-16 receptors , IL-17, IL-17 receptors, IL-18 (IGIF), IL-18 receptors, IL-1alpha, IL-1beta, IL-1 receptors, IL-2, IL-2 receptors, IL-20, IL- 20 receptors, IL-21, IL-21 receptors, IL-23, IL-23 receptors, IL-2 receptors, IL-3, IL-3 receptors, IL-31, IL-31 receptors, IL-3 receptors, IL -4, IL-4 receptors IL-5, IL-5 receptors, IL-6, IL-6 receptors, IL-7, IL-7 receptors, IL-8, IL-8 receptors, IL-9, IL-9 receptors, immunoglobulin immune complex, immunoglobulins, INF-alpha, INF-alpha receptors, INF-beta, INF-beta receptors, INF-gamma, INF-gamma receptors, IFN type-I, IFN type- I receptor, influenza, inhibin, Inhibin α, Inhibin β, iNOS, insulin, Insulin A-chain, Insulin B-chain, Insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding proteins, integrin , integrin alpha2, integrin alpha3, integrin alpha4, integrin alpha4 / beta1, integrin alpha-V / beta-3, integrin alpha-V / beta-6, integrin alpha4 / beta7, integrin alpha5 / beta1, integrin alpha5 / beta3, integrin alpha5 / beta6, integrin alphaσ (alphaV), integrin alphaθ, integrin beta1, integrin beta2, integrin beta3 (GPIIb-IIIa), IP-10, I-TAC, JE, kalliklein, Kallikrein 11, Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein L1, Kallikrein L2, Kallikrein L3, Kallikrein L4, kallistatin, KC, KDR, Keratinocyte Growth Factor (KGF), Keratinocyte Growth Facto r-2 (KGF-2), KGF, killer immunoglobulin-like receptor, kit ligand (KL), Kit tyrosine kinase, laminin 5, LAMP, LAPP (Amylin, islet-amyloid polypeptide), LAP (TGF-1), latency associated peptide, Latent TGF-1, Latent TGF-1 bp1, LBP, LDGF, LDL, LDL receptor, LECT2, Lefty, Leptin, leutinizing hormone (LH), Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, LFA-3 receptors, Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b, LTB4, LTBP-1, Lung surfactant, Luteinizing hormone, Lymphotactin, Lymphotoxin Beta Receptor, Lysosphingolipid receptor, Mac-1, macrophage-CSF (M-CSF), MAdCAM, MAG, MAP2, MARC, maspin, MCAM, MCK-2, MCP, MCP-1, MCP-2, MCP-3, MCP -4, MCP-I (MCAF), M-CSF, MDC, MDC (67 aa), MDC (69 aa), megsin, Mer, MET tyrosine kinase receptor family, METALLOPROTEASES, Membrane glycoprotein OX2, Mesothelin, MGDF receptor, MGMT , MHC (HLA-DR), microbial protein, MIF, MIG, MIP, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MIP-4, MK, MMAC1, MMP, MMP-1, MMP-10 , MMP-11, MMP-12, MMP- 13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, monocyte attractant protein, monocyte colony inhibitory factor, mouse gonadotropin-associated peptide, MPIF , Mpo, MSK, MSP, MUC-16, MUC18, mucin (Mud), Muellerian-inhibiting substance, Mug, MuSK, Myelin associated glycoprotein, myeloid progenitor inhibitor factor-1 (MPIF-I), NAIP, Nanobody, NAP, NAP -2, NCA 90, NCAD, N-Cadherin, NCAM, Neprilysin, Neural cell adhesion molecule, neroserpin, Neuronal growth factor (NGF), Neurotrophin-3, Neurotrophin-4, Neurotrophin-6, Neuropilin 1, Neurturin, NGF-beta , NGFR, NKG20, N-methionyl human growth hormone, nNOS, NO, Nogo-A, Nogo receptor, non-structural protein type 3 (NS3) from the hepatitis C virus, NOS, Npn, NRG-3, NT, NT- 3, NT-4, NTN, OB, OGG1, Oncostatin M, OP-2, OPG, OPN, OSM, OSM receptors, osteoinductive factors, osteopontin, OX40L, OX40R, oxidized LDL, p150, p95, PADPr, parathyroid hormone, PARC , PARP, PBR, PBSF, PCAD, P-Cadherin, PCNA, PCSK9, PDGF, PDGF receptor, PDGF-AA, PDG F-AB, PDGF-BB, PDGF-D, PDK-1, PECAM, PEDF, PEM, PF-4, PGE, PGF, PGI2, PGJ2, PIGF, PIN, PLA2, Placenta growth factor, placental alkaline phosphatase (PLAP) , placental lactogen, pl
asminogen activator inhibitor-1, platelet-growth factor, plgR, PLP, poly glycol chains of different size (eg PEG-20, PEG-30, PEG40), PP14, prekallikrein, prion protein, procalcitonin, Programmed cell death protein 1, proinsulin , prolactin, Proprotein convertase PC9, prorelaxin, prostate specific membrane antigen (PSMA), Protein A, Protein C, Protein D, Protein S, Protein Z, PS, PSA, PSCA, PsmAr, PTEN, PTHrp, Ptk, PTN, P- selectin glycoprotein ligand-1, R51, RAGE, RANK, RANKL, RANTES, relaxin, Relaxin A-chain, Relaxin B-chain, renin, respiratory syncytial virus (RSV) F, Ret, reticulon 4, Rheumatoid factors, RLI P76, RPA2 , RPK-1, RSK, RSV Fgp, S100, RON-8, SCF / KL, SCGF, Sclerostin, SDF-1, SDF1α, SDF1β, SERINE, Serum Amyloid P, Serum albumin, sFRP-3, Shh, Shiga like toxin II, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, sphingosine 1-phosphate receptor 1, Staphylococcal lipoteichoic acid, Stat, STEAP, STEAP-II, stem cell factor (SCF), streptokinase, superoxide dismutase, syndec an-1, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TB, TCA-3, T-cell receptor alpha / beta, TdT, TECK, TEM1, TEM5, TEM7, TEM8, Tenascin, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta Rl (ALK-5 ), TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, TGF-I, Thrombin, thrombopoietin (TPO), Thymic stromal lymphoprotein receptor, Thymus Ck-1, thyroid stimulating hormone (TSH), thyroxine, thyroxine-binding globulin, Tie, TIMP, TIQ, Tissue Factor, tissue factor protease inhibitor, tissue factor protein, TMEFF2, Tmpo, TMPRSS2, TNF receptor I, TNF receptor II, TNF-alpha, TNF-beta, TNF-beta2 , TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2 / DR4), TNFRSF10B (TRAIL R2 DR5 / KILLER / TRICK-2A / TRICK-B), TNFRSF10C (TRAIL R3 DcR1 / LIT / TRID), TNFRSF10D (TRAIL R4 DcR2 / TRUNDD), TNFRSF11A (RANK ODF R / TRANCE R), TNFRSF11B (OPG OCIF / TR1), TNFRSF12 (TWEAK R FN14), TNFRSF12A, TNFRSF13B (TACI ), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR / HveA / LIGHT R / TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ / TRADE), TNFRSF19L (RELT) , TNFRSF1A (TNF Rl CD120a / p55-60), TNFRSF1B (TNF RII CD120b / p75-80), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRSF25 (DR3 Apo-3 / LARD / TR-3 / TRAMP / WSL-1), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII / TNFC R), TNFRSF4 (OX40 ACT35 / TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1 / APT1 / CD95), TNFRSF6B (DcR3 M68 / TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1 BB CD137 / ILA), TNFRST23 (DcTRAIL R1 TNFRH1), TNFSF10 (TRAIL Apo-2 Ligand / TL2), TNFSF11 (TRANCE / RANK Ligand ODF / OPG Ligand), TNFSF12 (TWEAK Apo-3 Ligand / DR3 Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS / TALL1 / THANK / TNFSF20), TNFSF14 (LIGHT HVEM Ligand / LTg), TNFSF15 (TL1A / VEGI ), TNFSF18 (GITR Ligand AITR Ligand / TL6), TNFSF1A (TNF-a Conectin / DIF / TNFSF2), TNFSF1B (TNF-b LTa / TNFSF1), TNFSF3 (LTb TNFC / p33), TNFSF4 (OX40 Ligand gp34 / TXGP1) , TNFSF5 (CD40 Ligand CD154 / g p39 / HIGM1 / IMD3 / TRAP), TNFSF6 (Fas Ligand Apo-1 Ligand / APT1 Ligand), TNFSF7 (CD27 Ligand CD70), TNFSF8 (CD30 Ligand CD153), TNFSF9 (4-1 BB Ligand CD137 Ligand), TNF-α , TNF-β, TNIL-I, toxic metabolite, TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, transforming growth factors (TGF) such as TGF- alpha and TGF-beta, Transmembrane glycoprotein NMB, Transthyretin, TRF, Trk, TROP-2, Trophoblast glycoprotein, TSG, TSLP, Tumor Necrosis Factor (TNF), tumor-associated antigen CA 125, tumor-associated antigen expressing Lewis Y related carbohydrate , TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VAP-1, vascular endothelial growth factor (VEGF), vaspin, VCAM, VCAM-1, VECAD, VE-Cadherin, VE-Cadherin-2, VEFGR-1 (flt-1), VEFGR-2, VEGF receptor (VEGFR), VEGFR-3 (flt-4), VEGI, VIM, Viral antigens, VitB12 receptor, Vitronectin receptor, VLA, VLA-1, VLA-4, VNR integrin , von Willebrand Factor (vWF), WIF-1, WNT1, WNT10A, WNT10B, WNT11, WNT16, WNT2, WNT2B / 13, WNT3, WNT3A, WNT 4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1, XCL2 / SCM-l-beta, XCLl / Lymphotactin, XCR1, XEDAR, XIAP, XPD.
また、本発明の抗原結合分子に含まれる抗体の2つの可変領域のうち、もう一方の可変領域が結合する、先の「第1の抗原」と「第2の抗原」とは異なる「第3の抗原」としては、例えば、腫瘍細胞に特異的な抗原が好ましく、細胞の悪性化に伴って発現する抗原の他、細胞が、がん化した際に細胞表面やタンパク質分子上に現れる異常な糖鎖も含まれる。具体的には、例えば、ALK受容体(プレイオトロフィン受容体)、プレイオトロフィン、KS 1/4膵臓癌抗原、卵巣癌抗原(CA125)、前立腺酸リン酸、前立腺特異的抗原(PSA)、メラノーマ関連抗原p97、メラノーマ抗原gp75、高分子量メラノーマ抗原(HMW-MAA)、前立腺特異的膜抗原、癌性胚抗原(CEA)、多型上皮ムチン抗原、ヒト乳脂肪球抗原、CEA、TAG-72、CO17-1A、GICA 19-9、CTA-1およびLEAなどの結腸直腸腫瘍関連抗原、バーキットリンパ腫抗原-38.13、CD19、ヒトBリンパ腫抗原-CD20、CD33、ガングリオシドGD2、ガングリオシドGD3、ガングリオシドGM2およびガングリオシドGM3などのメラノーマ特異的抗原、腫瘍特異的移植型細胞表面抗原(TSTA)、T抗原、DNA腫瘍ウイルスおよびRNA腫瘍ウイルスのエンベロープ抗原などのウイルスにより誘導される腫瘍抗原、結腸のCEA、5T4癌胎児トロホブラスト糖タンパク質および膀胱腫瘍癌胎児抗原などの癌胎児抗原α-フェトプロテイン、ヒト肺癌抗原L6およびL20などの分化抗原、線維肉腫の抗原、ヒト白血病T細胞抗原-Gp37、新生糖タンパク質、スフィンゴ脂質、EGFR(上皮増殖因子受容体)などの乳癌抗原、NY-BR-16、NY-BR-16およびHER2抗原(p185HER2)、多型上皮ムチン(PEM)、悪性ヒトリンパ球抗原-APO-1、胎児赤血球に認められるI抗原などの分化抗原、成人赤血球に認められる初期内胚葉I抗原、移植前の胚、胃癌に認められるI(Ma)、乳腺上皮に認められるM18、M39、骨髄細胞に認められるSSEA-1、VEP8、VEP9、Myl、VIM-D5、結腸直腸癌に認められるD156-22、TRA-1-85(血液群H)、精巣および卵巣癌に認められるSCP-1、結腸癌に認められるC14、肺癌に認められるF3、胃癌に認められるAH6、Yハプテン、胚性癌細胞に認められるLey、TL5(血液群A)、A431細胞に認められるEGF受容体、膵臓癌に認められるE1シリーズ(血液群B)、胚性癌細胞に認められるFC10.2、胃癌抗原、腺癌に認められるCO-514(血液群Lea)、腺癌に認められるNS-10、CO-43(血液群Leb)、A431細胞のEGF受容体に認められるG49、結腸癌に認められるMH2(血液群ALeb/Ley)、結腸癌に認められる19.9、胃癌ムチン、骨髄細胞に認められるT5A7、メラノーマに認められるR24、胚性癌細胞に認められる4.2、GD3、D1.1、OFA-1、GM2、OFA-2、GD2、およびM1:22:25:8ならびに4~8細胞段階の胚に認められるSSEA-3およびSSEA-4、皮下T細胞リンパ腫抗原、MART-1抗原、シアリルTn(STn)抗原、結腸癌抗原NY-CO-45、肺癌抗原NY-LU-12変異体A、腺癌抗原ART1、腫瘍随伴性関連脳-精巣癌抗原(癌神経抗原MA2、腫瘍随伴性神経抗原)、神経癌腹部抗原2(NOVA2)、血液細胞癌抗原遺伝子520、腫瘍関連抗原CO-029、腫瘍関連抗原MAGE-C1(癌/精巣抗原CT7)、MAGE-B1(MAGE-XP抗原)、MAGE-B2(DAM6)、MAGE-2、MAGE-4a、MAGE-4bおよびMAGE-X2、癌-精巣抗原(NY-EOS-1)、YKL-40および上記ポリペプチドのいずれかの断片またはこれらに対して修飾された構造等(前記の修飾リン酸基や糖鎖等)、EpCAM、EREG、CA19-9、CA15-3、シリアルSSEA-1(SLX)、HER2、PSMA、CEA、CLEC12A等が挙げられる。 Among the two variable regions of an antibody contained in the antigen-binding molecule of the present invention, a “first antigen to which a variable region that can bind to two different antigens but cannot simultaneously bind to these antigens binds. "And" second antigen "include, for example, immune cell surface molecules (eg, T cell surface molecules, NK cell surface molecules, dendritic cell surface molecules, B cell surface molecules, NKT cell surface molecules, MDSC cell surface molecules Antigens (integrin, tissue factor, VEGFR, PDGFR, EGFR, IGFR, MET chemokine receptor, heparan) expressed in normal cells, tumor cells, tumor blood vessels, stromal cells, etc. Proteoglycan sulfate, CD44, fibronectin, DR5, TNFRSF, etc.) are preferred, and the combination of “first antigen” and “second antigen” is any of the first antigen and the second antigen. One of them is, for example, a molecule that is specifically expressed on T cells, and the other antigen is preferably a molecule that is expressed on the surface of T cells or other immune cells. In another embodiment, the combination of the “first antigen” and the “second antigen” includes any one of the first antigen and the second antigen expressed specifically on T cells, for example. Preferably, the other antigen is a molecule that is expressed on immune cells and is different from the previously selected antigen. Specifically, for example, CD3 and a T cell receptor can be mentioned as molecules specifically expressed on T cells. CD3 is particularly preferable. As the CD3 site to which the antigen-binding molecule of the present invention binds, for example, in the case of human CD3, it binds to any epitope as long as it exists in the γ chain, δ chain or ε chain sequence constituting human CD3 It may be a thing. In particular, an epitope present in the extracellular region of the ε chain of the human CD3 complex is preferred. The structure of the γ chain, δ chain, or ε chain constituting CD3 is such that the polynucleotide sequence is SEQ ID NO: 83 (NM_000073.2), 85 (NM_000732.4) and 87 (NM_000733.3). The sequences are described in SEQ ID NOs: 84 (NP_000064.1), 86 (NP_000723.1) and 88 (NP_000724.1) (in parentheses indicate RefSeq registration numbers). Still other antigens include Fcγ receptor, TLR, lectin, IgA, immune checkpoint molecule, TNF superfamily molecule, TNFR superfamily molecule, and NK receptor molecule.
In addition, the “first antigen” and the “second antigen” to which the other variable region of the two variable regions of the antibody contained in the antigen-binding molecule of the present invention binds are different from the “third antigen”. For example, antigens specific to tumor cells are preferable. In addition to antigens expressed as cells become malignant, abnormalities appearing on the cell surface or protein molecules when cells become cancerous. Sugar chains are also included. Specifically, for example, ALK receptor (pleiotrophin receptor), pleiotrophin, KS 1/4 pancreatic cancer antigen, ovarian cancer antigen (CA125), prostatic acid phosphate, prostate specific antigen (PSA), Melanoma-associated antigen p97, melanoma antigen gp75, high molecular weight melanoma antigen (HMW-MAA), prostate specific membrane antigen, cancerous embryo antigen (CEA), polymorphic epithelial mucin antigen, human milk fat globule antigen, CEA, TAG-72 , CO17-1A, GICA 19-9, CTA-1 and LEA and other colorectal tumor associated antigens, Burkitt lymphoma antigen-38.13, CD19, human B lymphoma antigen-CD20, CD33, ganglioside GD2, ganglioside GD3, ganglioside GM2 and Tumor antigens induced by viruses such as melanoma-specific antigens such as ganglioside GM3, tumor-specific transplanted cell surface antigen (TSTA), T antigen, DNA tumor virus and RNA tumor virus envelope antigen, colon CEA, carcinoembryonic antigen α-fetoprotein such as 5T4 carcinoembryonic trophoblast glycoprotein and bladder tumor carcinoembryonic antigen, differentiation antigens such as human lung cancer antigens L6 and L20, fibrosarcoma antigen, human leukemia T cell antigen-Gp37, nascent glycoprotein , Sphingolipids, breast cancer antigens such as EGFR (epidermal growth factor receptor), NY-BR-16, NY-BR-16 and HER2 antigen (p185HER2), polymorphic epithelial mucin (PEM), malignant human lymphocyte antigen-APO- 1. Differentiation antigens such as I antigen found in fetal erythrocytes, early endoderm I antigen found in adult erythrocytes, pre-implantation embryo, I (Ma) found in gastric cancer, M18, M39, bone marrow cells found in mammary epithelium SSEA-1, VEP8, VEP9, Myl, VIM-D5, D156-22 found in colorectal cancer, TRA-1-85 (blood group H), SCP-1 found in testis and ovarian cancer, colon C14 found in cancer, F3 found in lung cancer, AH6 found in stomach cancer, Y hapten, Ley found in embryonic cancer cells, TL5 (blood group A), EGF receptor found in A431 cells, E1 series found in pancreatic cancer (blood group B), FC10 found in embryonic cancer cells. 2. Gastric cancer antigen, CO-514 (blood group Lea) found in adenocarcinoma, NS-10, CO-43 (blood group Leb) found in adenocarcinoma, G49 found in EGF receptor of A431 cells, colon cancer MH2 (blood group ALeb / Ley), colon cancer 19.9, gastric cancer mucin, T5A7 found in bone marrow cells, R24 found in melanoma, 4.2 found in embryonic cancer cells, GD3, D1.1, OFA-1, GM2, OFA-2, GD2, and M1: 22: 25: 8 and SSEA-3 and SSEA-4, subcutaneous T-cell lymphoma antigen, MART-1 antigen found in 4-8 cell stage embryos, Sialyl Tn (STn) antigen, colon cancer antigen NY-CO-45, lung cancer antigen NY-LU-12 variant A, adenocarcinoma antigen ART1, tumor-associated brain-testis cancer antigen (cancer neuronal antigen MA2, tumor-associated Nerve antigen) , Neuronal cancer abdominal antigen 2 (NOVA2), blood cell cancer antigen gene 520, tumor-associated antigen CO-029, tumor-associated antigen MAGE-C1 (cancer / testis antigen CT7), MAGE-B1 (MAGE-XP antigen), MAGE- B2 (DAM6), MAGE-2, MAGE-4a, MAGE-4b and MAGE-X2, cancer-testis antigen (NY-EOS-1), YKL-40 and any fragment of the above polypeptides or against these Examples include modified structures (such as the above-mentioned modified phosphate groups and sugar chains), EpCAM, EREG, CA19-9, CA15-3, serial SSEA-1 (SLX), HER2, PSMA, CEA, CLEC12A, and the like.
(1)哺乳類細胞、:CHO(Chinese hamster ovary cell line)、COS(Monkey kidney cell line)、ミエローマ(Sp2/O、NS0等)、BHK (baby hamster kidney cell line)、HEK293(human embryonic kidney cell line with sheared adenovirus (Ad)5 DNA)、PER.C6 cell (human embryonic retinal cell line transformed with the Adenovirus Type 5 (Ad5) E1A and E1B genes)、Hela、Vero、など(Current Protocols in Protein Science (May, 2001, Unit 5.9, Table 5.9.1))
(2)両生類細胞:アフリカツメガエル卵母細胞など
(3)昆虫細胞:sf9、sf21、Tn5など
また、抗体は大腸菌(mAbs 2012 Mar-Apr; 4(2): 217-225.)や酵母(WO2000023579)でも作製することができる。大腸菌で作製した抗体は糖鎖が付加されていない。一方、酵母で作製した抗体は糖鎖が付加される。 The antigen-binding molecule of the present invention can be produced by methods known to those skilled in the art. For example, the antibody can be prepared by the following method, but is not limited thereto. Many combinations of host cells and expression vectors for producing antibodies by introducing a gene encoding an isolated polypeptide into a suitable host are known. Any of these expression systems can be applied to isolate the antigen-binding molecule of the present invention. When eukaryotic cells are used as host cells, animal cells, plant cells, or fungal cells can be used as appropriate. Specifically, the following cells can be exemplified as animal cells.
(1) Mammalian cells: CHO (Chinese hamster ovary cell line), COS (Monkey kidney cell line), myeloma (Sp2 / O, NS0, etc.), BHK (baby hamster kidney cell line), HEK293 (human embryonic kidney cell line) with sheared adenovirus (Ad) 5 DNA), PER.C6 cell (human embryonic retinal cell line transformed with the Adenovirus Type 5 (Ad5) E1A and E1B genes), Hela, Vero, etc. (Current Protocols in Protein Science (May, 2001 , Unit 5.9, Table 5.9.1))
(2) Amphibian cells: Xenopus oocytes, etc. (3) Insect cells: sf9, sf21, Tn5, etc. In addition, antibodies are used in Escherichia coli (mAbs 2012 Mar-Apr; 4 (2): 217-225.) And yeast (WO2000023579). ). The antibody produced in E. coli has no sugar chain added. On the other hand, sugar chains are added to antibodies produced in yeast.
またアミノ酸改変は置換に限られず、欠失、付加、挿入、又は修飾のいずれか、又はそれらの組み合わせであってもよい。 In addition, a DNA encoding a protein in which one or a plurality of amino acid residues in the variable region of an antibody prepared using a known method for an antigen is substituted with another amino acid of interest, By chemically synthesizing the DNA, it is possible to obtain DNA encoding a heavy chain in which one or more amino acid residues in the variable region are substituted with other amino acids of interest. The amino acid substitution site and the type of substitution are not particularly limited. Preferred regions for amino acid modification include regions exposed to solvent in the variable region and loop regions. Of these, CDR1, CDR2, CDR3, FR3 region and loop region are preferable. Specifically, Kabat numbering 31 to 35, 50 to 65, 71 to 74, 95 to 102 of the heavy chain variable region, Kabat numbering 24 to 34, 50 to 56, 89 to 97 of the light chain variable region are preferable. More preferred are Kabat numbering 31, 52a-61, 71-74, 97-101 of the chain variable region, and Kabat numbering 24-34, 51-56, 89-96 of the L chain variable region.
The amino acid modification is not limited to substitution, and may be any one of deletion, addition, insertion, modification, or a combination thereof.
(i)第1の抗原又は第2の抗原に結合する抗体の可変領域の少なくとも1つのアミノ酸が改変された抗原結合分子であって、該改変された可変領域のアミノ酸の少なくとも1つが互いに異なる可変領域を含む抗原結合分子のライブラリーを作製する工程、
(ii)作製されたライブラリーの中から、第1の抗原及び第2の抗原に対して結合活性を有するが、当該第1の抗原及び第2の抗原と同時には結合しない可変領域を含む抗原結合分子を選択する工程、
(iii)工程(ii)で選択された抗原結合分子の該可変領域をコードする核酸と、第3の抗原に結合する抗原結合分子の可変領域をコードする核酸とを含む宿主細胞を培養して、第1の抗原と第2の抗原に結合することができるが該第1の抗原と第2の抗原とが同時には結合しない抗体の可変領域、及び、第3の抗原に結合する可変領域を含む、抗原結合分子を発現させる工程、並びに
(iv)前記宿主細胞培養物から抗原結合分子を回収する工程。 For example, a production method including the following steps can be mentioned:
(i) an antigen-binding molecule in which at least one amino acid of a variable region of an antibody that binds to the first antigen or the second antigen is modified, wherein at least one of the amino acids of the modified variable region is different from each other Creating a library of antigen binding molecules comprising the region,
(ii) an antigen comprising a variable region that has binding activity to the first antigen and the second antigen from the prepared library, but does not bind simultaneously with the first antigen and the second antigen; Selecting a binding molecule,
(iii) culturing a host cell containing a nucleic acid encoding the variable region of the antigen-binding molecule selected in step (ii) and a nucleic acid encoding the variable region of the antigen-binding molecule that binds to the third antigen. A variable region of an antibody that can bind to the first antigen and the second antigen but does not bind to the first antigen and the second antigen at the same time, and a variable region that binds to the third antigen. Comprising the step of expressing an antigen binding molecule, and
(iv) recovering the antigen-binding molecule from the host cell culture.
(v) 作製されたライブラリーの中から、第1の抗原及び第2の抗原に対して結合活性を有するが、それぞれ異なる細胞上で発現している第1の抗原と第2の抗原に同時には結合しない可変領域を含む抗原結合分子を選択する工程。 In this production method, step (ii) may be the following selection step:
(v) Among the prepared libraries, the first antigen and the second antigen have binding activity to the first antigen and the second antigen, but are expressed on different cells, respectively. Selecting an antigen-binding molecule comprising a variable region that does not bind.
複数組み合わせて使用する場合、組み合わせる数は特に限定されず、例えば、2個以上30個以下、好ましくは2個以上25個以下、2個以上22個以下、2個以上20個以下、2個以上15個以下、2個以上10個以下、2個以上5個以下、2個以上3個以下である。
複数組み合わせる場合、抗体の重鎖可変領域又は軽鎖可変領域のみに当該アミノ酸改変を加えてもよく、重鎖可変領域と軽鎖可変領域の双方に適宜振り分けて加えてもよい。 The amino acid modification of the present invention may be used alone or in combination.
When used in combination, the number of combinations is not particularly limited. For example, 2 or more and 30 or less, preferably 2 or more and 25 or less, 2 or more and 22 or less, 2 or more and 20 or less, 2 or more 15 or less, 2 or more and 10 or less, 2 or more and 5 or less, 2 or more and 3 or less.
In the case of combining a plurality, the amino acid modification may be added only to the heavy chain variable region or the light chain variable region of the antibody, or may be appropriately distributed to both the heavy chain variable region and the light chain variable region.
(i)第1の抗原又は第2の抗原に結合する抗体の可変領域の少なくとも1つのアミノ酸が改変された抗原結合分子であって、該改変された可変領域のアミノ酸の少なくとも1つが互いに異なる可変領域を含む抗原結合分子のライブラリーを作製する工程、
(ii)作製されたライブラリーの中から、第1の抗原及び第2の抗原に対して結合活性を有するが、当該第1の抗原及び第2の抗原と同時には結合しない可変領域を含む抗原結合分子を選択する工程、
(iii)工程(ii)で選択された抗原結合分子の該可変領域をコードする核酸を含む宿主細胞を培養して、第1の抗原と第2の抗原に結合することができるが該第1の抗原と第2の抗原とが同時には結合しない抗体の可変領域を含む、抗原結合分子を発現させる工程、並びに
(iv)前記宿主細胞培養物から抗原結合分子を回収する工程。
なお、上記アミノ酸改変のための好ましい領域としては、重鎖可変領域が挙げられる。さらに好ましくは可変領域中の溶媒に露出している領域およびループ領域が挙げられる。中でも、CDR1、CDR2、CDR3、FR3領域、ループ領域が好ましい。具体的には、H鎖可変領域のKabatナンバリング31~35、50~65、71~74、95~102、L鎖可変領域のKabatナンバリング24~34、50~56、89~97が好ましく、H鎖可変領域のKabatナンバリング31、52a~61、71~74、97~101、L鎖可変領域のKabatナンバリング24~34、51~56、89~96がより好ましい。 Examples of the method for producing such an antigen-binding molecule include a production method including the following steps:
(i) an antigen-binding molecule in which at least one amino acid of a variable region of an antibody that binds to the first antigen or the second antigen is modified, wherein at least one of the amino acids of the modified variable region is different from each other Creating a library of antigen binding molecules comprising the region,
(ii) an antigen comprising a variable region that has binding activity to the first antigen and the second antigen from the prepared library, but does not bind simultaneously with the first antigen and the second antigen; Selecting a binding molecule,
(iii) A host cell containing a nucleic acid encoding the variable region of the antigen-binding molecule selected in step (ii) can be cultured to bind to the first and second antigens. Expressing an antigen-binding molecule comprising a variable region of an antibody that does not bind to the antigen and the second antigen simultaneously, and
(iv) recovering the antigen-binding molecule from the host cell culture.
A preferred region for the amino acid modification includes a heavy chain variable region. More preferably, a region exposed to the solvent in the variable region and a loop region are included. Of these, CDR1, CDR2, CDR3, FR3 region and loop region are preferable. Specifically, Kabat numbering 31 to 35, 50 to 65, 71 to 74, 95 to 102 of the heavy chain variable region, Kabat numbering 24 to 34, 50 to 56, 89 to 97 of the light chain variable region are preferable. More preferred are Kabat numbering 31, 52a-61, 71-74, 97-101 of the chain variable region, and Kabat numbering 24-34, 51-56, 89-96 of the L chain variable region.
(v) 作製されたライブラリーの中から、第1の抗原及び第2の抗原に対して結合活性を有するが、それぞれ異なる細胞上で発現している第1の抗原と第2の抗原に同時には結合しない可変領域を含む抗原結合分子を選択する工程。 In the present production method, the step (ii) may be the following selection step:
(v) Among the prepared libraries, the first antigen and the second antigen have binding activity to the first antigen and the second antigen, but are expressed on different cells, respectively. Selecting an antigen-binding molecule comprising a variable region that does not bind.
複数組み合わせて使用する場合、組み合わせる数は特に限定されず、例えば、2個以上30個以下、好ましくは2個以上25個以下、2個以上22個以下、2個以上20個以下、2個以上15個以下、2個以上10個以下、2個以上5個以下、2個以上3個以下である。
複数組み合わせる場合、抗体の重鎖可変領域又は軽鎖可変領域のいずれか一方のみに当該アミノ酸改変を加えてもよく、重鎖可変領域と軽鎖可変領域の双方に適宜振り分けて加えてもよい。 The amino acid modification of the present invention may be used alone or in combination.
When used in combination, the number of combinations is not particularly limited. For example, 2 or more and 30 or less, preferably 2 or more and 25 or less, 2 or more and 22 or less, 2 or more and 20 or less, 2 or more 15 or less, 2 or more and 10 or less, 2 or more and 5 or less, 2 or more and 3 or less.
When combining two or more, the amino acid modification may be added to either one of the heavy chain variable region or the light chain variable region of the antibody, or may be appropriately distributed and added to both the heavy chain variable region and the light chain variable region.
本方法によって導入されるアミノ酸改変の種類や範囲は特に限定されるものではない。 Furthermore, antigen-binding molecules produced by the production method are also included in the present invention.
The type and range of amino acid modification introduced by this method is not particularly limited.
1. 第1の抗原に結合する抗原結合分子に第2の抗原に結合するペプチド(この用語はポリペプチドおよびタンパク質を含むようにして用いられる)を挿入する方法
2. 抗原結合分子中のループを長く改変(延長)することができる位置に、様々なアミノ酸が出現するようなライブラリを作製して、任意の第2の抗原に対して結合活性を有する抗原結合分子を、ライブラリから抗原への結合活性を指標に取得する方法
3. あらかじめ第一の抗原に対して結合することが知られている抗原結合分子から、部位特異的変異法によって作製した抗体を用いて、第一の抗原との結合活性を維持するアミノ酸を同定し、同定されたアミノ酸が出現するようなライブラリから、任意の第2の抗原に対して結合活性を有する抗原結合分子を、抗原への結合活性を指標に取得する方法
4. 3の方法において、さらに、抗原結合分子中のループを長く改変(延長)することができる位置に様々なアミノ酸が出現するような抗体ライブラリを作製して、任意の第2の抗原に対して結合活性を有する抗原結合分子を、ライブラリから抗原への結合活性を指標に取得する方法
5. 1.2.3.又は4.の方法において、糖鎖付加配列(例えばNxS, NxT、xはP以外のアミノ酸)が出現するように改変し、糖鎖レセプターが認識する糖鎖を付加させる方法(例えばハイマンノース型糖鎖を付加し、ハイマンノースレセプターが認識する。ハイマンノース型糖鎖は抗体発現時にキフネンシンを添加することで得られることが知られている(MAbs. 2012 Jul-Aug;4(4):475-87))
6. 1.2.3.又は4.の方法において、ループ部位や各種アミノ酸へ改変することが可能であった部位にCys、Lysもしくは非天然アミノ酸を挿入または置換して、第2の抗原に結合するドメインを共有結合で付加する方法(Antibody drug conjugateに代表される方法であり、Cys、Lysもしくは非天然アミノ酸へ共有結合で結合させる方法(mAbs 6:1, 34-45; January/February 2014、WO2009/134891A2、Bioconjug Chem. 2014 Feb 19;25(2):351-61))
なお、上記に例示された6つのライブラリ作製方法において、抗原結合分子中のアミノ酸を置換する箇所又は抗原結合分子中にペプチドを挿入する箇所は、抗原結合分子のFab又は可変領域部分が好ましい。好ましい領域としては、可変領域中の溶媒に露出している領域およびループ領域が挙げられる。中でも、CDR1、CDR2、CDR3、FR3領域、ループ領域が好ましい。具体的には、H鎖可変領域のKabatナンバリング31~35、50~65、71~74、95~102、L鎖可変領域のKabatナンバリング24~34、50~56、89~97が好ましく、H鎖可変領域のKabatナンバリング31、52a~61、71~74、97~101、L鎖可変領域のKabatナンバリング24~34、51~56、89~96がより好ましい。 As one non-limiting aspect of the present invention, the following six methods are exemplified for preparing a library.
1. 1. Inserting a peptide that binds to a second antigen (this term is used to include polypeptides and proteins) into an antigen-binding molecule that binds to the first antigen. A library in which various amino acids appear at a position where a loop in the antigen-binding molecule can be modified (extended) long can be prepared, and an antigen-binding molecule having binding activity against any second antigen can be obtained. 2. A method for obtaining a binding activity to an antigen from a library as an
6). 1.2.3. Alternatively, in the method of 4., Cys, Lys, or a non-natural amino acid is inserted or substituted at a loop site or a site that could be modified to various amino acids, and a domain that binds to the second antigen is added by a covalent bond. (Methods represented by antibody drug conjugates, such as covalent coupling to Cys, Lys or unnatural amino acids (mAbs 6: 1, 34-45; January / February 2014, WO2009 / 134891A2, Bioconjug Chem. 2014
In the six library preparation methods exemplified above, Fab or a variable region part of the antigen-binding molecule is preferably used as the position where the amino acid in the antigen-binding molecule is substituted or the position where the peptide is inserted into the antigen-binding molecule. Preferred regions include regions exposed to solvent in the variable region and loop regions. Of these, CDR1, CDR2, CDR3, FR3 region and loop region are preferable. Specifically, Kabat numbering 31 to 35, 50 to 65, 71 to 74, 95 to 102 of the heavy chain variable region, Kabat numbering 24 to 34, 50 to 56, 89 to 97 of the light chain variable region are preferable. More preferred are Kabat numbering 31, 52a-61, 71-74, 97-101 of the chain variable region, and Kabat numbering 24-34, 51-56, 89-96 of the L chain variable region.
ステップ1:CD3結合能が保持されているアミノ酸を選択(CD3結合量が未改変抗体の80%以上であること)
例えば、ステップ1で選択されたアミノ酸が出現するように、CD3と第2の抗原に結合する抗体取得のためのライブラリを作製することができる。 As one non-limiting embodiment of the present invention, an antibody library for obtaining an antibody that binds to CD3 and a second antigen can be designed as follows.
Step 1: Select amino acids that retain CD3 binding ability (CD3 binding amount should be 80% or more of unmodified antibody)
For example, a library for obtaining an antibody that binds to CD3 and the second antigen can be prepared so that the amino acid selected in
ステップ1:CD3結合能が保持されているアミノ酸を選択(CD3結合量が未改変抗体の80%以上であること)
ステップ2:H鎖CDR3の99-100(Kabat numbering)の間にアミノ酸を挿入
例えば、ステップ1に加えてステップ2でCDR3領域にアミノ酸を挿入することにより、ライブラリの多様性を増強させた、CD3と第2の抗原に結合する抗体取得のためのライブラリを作製することができる。 As one non-limiting embodiment of the present invention, an antibody library for obtaining an antibody that binds to CD3 and a second antigen can be designed as follows.
Step 1: Select amino acids that retain CD3 binding ability (CD3 binding amount should be 80% or more of unmodified antibody)
Step 2: Insert amino acids between 99-100 (Kabat numbering) of heavy chain CDR3 For example, CD3 was enhanced by inserting amino acids into the CDR3 region in
ステップ1:CD3結合能が保持されているアミノ酸を選択(CD3結合量が未改変抗体の80%以上であること)
ステップ2:ECM結合が改変前よりもMRAと比較して10倍以内であるアミノ酸を選択
ステップ3:H鎖CDR3の99-100(Kabat numbering)の間にアミノ酸挿入
例えば、ステップ1及び3に加えて、ステップ2を加えることで、ライブラリで出現するアミノ酸についても、ECM結合が増強されないアミノ酸を選択することができるが、この手法に限定されることはない。また、ステップ2を経ないライブラリ設計であっても、ライブラリから取得された抗原結合分子に対してECM結合を測定し、評価することが可能である。 As one non-limiting embodiment of the present invention, an antibody library for obtaining an antibody that binds to CD3 and a second antigen can be designed as follows.
Step 1: Select amino acids that retain CD3 binding ability (CD3 binding amount should be 80% or more of unmodified antibody)
Step 2: Select amino acids whose ECM binding is within 10 times compared to MRA than before modification Step 3: Insert amino acids between 99-100 (Kabat numbering) of heavy chain CDR3 For example, in addition to
VH領域CE115HA000(配列番号:52)に、V11L/L78Iのアミノ酸改変を加えた上記ライブラリが好ましいがこれに限定されることはない。さらに、VH領域CE115HA000(配列番号:52)に、V11L/D72A/L78I/D101Qのアミノ酸改変を加えた上記ライブラリが好ましいがこれに限定されることはない。 As a non-limiting embodiment of the present invention, when the VH region CE115HA000 (SEQ ID NO: 52) is used as a template sequence of a CD3 (CD3ε) -binding antibody, amino acids used for library design are included in the heavy chain variable region. Kabat numbering 11th, 31st, 52a, 52b, 52b, 52c, 53, 54, 56, 57, 61, 72, 78, 98, 99, 100, 100a , 100b-position, 100c-position, 100d-position, 100e-position, 100f-position, 100g-position, 101-position, any one or more amino acids, etc. can be exemplified.
The above library in which the VH region CE115HA000 (SEQ ID NO: 52) is modified with the amino acid modification of V11L / L78I is preferable, but is not limited thereto. Furthermore, the above-mentioned library in which the amino acid modification of V11L / D72A / L78I / D101Q is added to the VH region CE115HA000 (SEQ ID NO: 52) is preferable, but not limited thereto.
免疫グロブリンが、2分子以上の活性型FcγRへ同時に結合する、あるいは、別の抗原と活性型FcγRへ同時に結合することによって、活性化FcγRの架橋反応が起こると、FcγRのITAMシグナルが伝達され、免疫細胞の活性化が起こる可能性が考えられる。IgG型の抗体1分子は、上記の通り、1分子のFcγRにしか結合できないため、抗原存在下でのみ、2分子以上の活性型FcγRが架橋され、免疫細胞の活性化が起こる。 [Example 1] Binding to CD3 (first antigen) and another antigen (second antigen), and simultaneously to CD3 (first antigen) and another antigen (second antigen) on different cells Concept of modified immunoglobulin variable (Fab) region that does not bind to immunoglobulins Crosslinks of activated FcγR by simultaneously binding to two or more molecules of active FcγR, or simultaneously binding to another antigen and active FcγR When the reaction takes place, it is considered that the FcγR ITAM signal is transmitted and immune cells may be activated. As described above, one molecule of IgG-type antibody can bind only to one molecule of FcγR, and therefore, only in the presence of an antigen, two or more molecules of active FcγR are cross-linked to activate immune cells.
このようなDual Binding Fabの性質を利用すれば、例えば抗体を介してT細胞をリダイレクトすることにより癌抗原を発現している癌細胞を傷害させる技術に、さらに癌組織中インテグリンへのターゲット機能を持たせることにより、より癌特異性を高めることが可能となる。 Therefore, as a method for controlling such an unfavorable cross-linking reaction, in one variable (Fab) region, a part of the variable (Fab) region binds to the first antigen and does not participate in the binding. Thus, a variable binding region (Dual Binding Fab) binding to the second antigen was considered (FIG. 1). At this time, as shown in FIG. 1, when two adjacent portions in one variable (Fab) region are essential for binding to each antigen, the binding of the second antigen occurs when the first antigen binds. Is inhibited, and similarly, when the binding of the second antigen is bound, the binding of the first antigen is inhibited. Therefore, since such an improved antibody having the properties of Dual Binding Fab cannot simultaneously bind to the first antigen and the second antigen, the cross-linking reaction between the first antigen and the second antigen does not occur. It was thought that it was not (Fig. 2). In addition, when the first antigen and the second antigen are not expressed on the cell membrane as in the soluble protein, or both are present on the same cell, the first antigen and the second antigen Although it can bind to both antigens at the same time, when it is expressed on different cells, it does not bind at the same time, and the case where two cells are not cross-linked is also considered as a Dual Binding Fab (FIG. 3). On the other hand, it is considered that the antigen (third antigen) that binds to the other variable (Fab) region undergoes a crosslinking reaction with the first antigen (FIG. 4) and also with the second antigen. (FIG. 5). As the constant region of the antibody, an Fc region that binds to FcγR can be used, or an Fc region with reduced binding activity to FcγR can be used.
By utilizing such a property of Dual Binding Fab, for example, a technique for damaging cancer cells expressing cancer antigens by redirecting T cells via antibodies, and further a target function for integrin in cancer tissue. By having it, it becomes possible to further increase the cancer specificity.
1.第1の抗原に対する結合活性を有する
2.第2の抗原に対する結合活性を有する
3.第1の抗原及び第2の抗原に同時には結合しない
なお、「第1の抗原及び第2の抗原に同時には結合しない」には、第1の抗原が発現している細胞と第2の抗原が発現している細胞の2つの細胞を架橋しない、あるいは、それぞれ別々の細胞に発現している第1の抗原と第2の抗原に同時に結合しないこと、さらに、第1の抗原と第2の抗原が、可溶型タンパクのように細胞膜上に発現していない、或いは、両方が同一細胞上に存在する場合には、第1の抗原と第2の抗原の両方に同時に結合することができるが、それぞれ異なる細胞上で発現している場合には同時には結合することができない場合も含まれる。 That is, an antibody having the action shown in FIG. 1 can be created if the following properties can be imparted by improving the variable (Fab) region to form a dual binding Fab.
1. 1. It has binding activity for the first antigen. 2. It has binding activity for the second antigen. It does not bind to the first antigen and the second antigen at the same time. “Does not bind to the first antigen and the second antigen at the same time” means that the cell expressing the first antigen and the second antigen Do not cross-link two of the cells in which are expressed, or do not simultaneously bind to the first and second antigens expressed in separate cells, respectively, and If the antigen is not expressed on the cell membrane like a soluble protein, or both are present on the same cell, they can bind to both the first and second antigens simultaneously. However, when they are expressed on different cells, they may not be able to bind at the same time.
1.T細胞上の第1の抗原に対する結合活性を有する
2.抗原提示細胞上の第2の抗原に対する結合活性を有する
3.第1の抗原及び第2の抗原に同時には結合しない Similarly, by modifying the variable (Fab) region to form a dual binding Fab, if the following properties can be imparted, for example, an antibody having the action shown in FIG. 6 can be created. is there.
1. 1. has binding activity to the first antigen on
(2-1)ヒトCD3、カニクイザルCD3発現細胞免疫ラットを用いたハイブリドーマの作製
SDラット(雌、免疫開始時6週齢、日本チャールス・リバー)に、ヒトCD3εγまたはカニクイザルCD3εγ発現Ba/F3細胞を以下の通り免疫した。初回免疫時を0日目とすると、0日目にフロイント完全アジュバント(Difco)とともに、5 x 107個のヒトCD3εγ発現Ba/F3細胞を腹腔内投与した。14日目にフロイント不完全アジュバント(Difco)とともに5 x 107個のカニクイザルCD3εγ発現Ba/F3細胞を腹腔内投与し、その後、1週間おきに4回5 x 107個のヒトまたはカニクイザルCD3εγ発現Ba/F3細胞を交互に腹腔内投与した。CD3εγの最終投与1週間後に(49日目)、ブーストとしてヒトCD3εγ発現Ba/F3細胞を静脈内投与し、その3日後に、ラットの脾臓細胞とマウスミエローマ細胞SP2/0とを、PEG1500(Roche Diagnostics)を用いた常法に従い細胞融合した。融合細胞、すなわちハイブリドーマは、10% FBSを含むRPMI1640培地 (以下、10%FBS/RPMI1640と称す)にて培養した。 [Example 2] Preparation of anti-human, cynomolgus monkey CD3ε antibody CE115 (2-1) Preparation of hybridoma using human CD3, cynomolgus monkey CD3-expressing cell-immunized rat SD rat (female, 6 weeks old at the start of immunization, Charles River, Japan) ) Were immunized with human CD3εγ or cynomolgus monkey CD3εγ-expressing Ba / F3 cells as follows. When the first immunization was taken as
ハイブリドーマ細胞から、RNeasy Mini Kits(QIAGEN)を用いてトータルRNAを抽出し、SMART RACE cDNA Amplification Kit(BD Biosciences)によりcDNAを合成した。作製したcDNAを用いて、PCRにより、抗体の可変領域遺伝子をクローニングベクターに挿入した。各DNA断片の塩基配列は、BigDye Terminator Cycle Sequencing Kit(Applied Biosystems)を用い、DNAシークエンサーABI PRISM 3700 DNA Sequencer(Applied Biosystems)にて、添付説明書記載の方法に従い決定した。CE115 H鎖可変領域(配列番号:13)及びCE115 L鎖可変領域(配列番号:14)のCDR、FRの決定はKabat numberingに従って行った。 (2-2) Preparation of anti-human, monkey CD3ε chimeric antibody Total RNA was extracted from hybridoma cells using RNeasy Mini Kits (QIAGEN), and cDNA was synthesized using SMART RACE cDNA Amplification Kit (BD Biosciences). Using the prepared cDNA, the antibody variable region gene was inserted into a cloning vector by PCR. The base sequence of each DNA fragment was determined using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) with the DNA sequencer ABI PRISM 3700 DNA Sequencer (Applied Biosystems) according to the method described in the attached instructions. The determination of CDR and FR of CE115 heavy chain variable region (SEQ ID NO: 13) and CE115 light chain variable region (SEQ ID NO: 14) was performed according to Kabat numbering.
次に、癌抗原(EGFR)に対するIgGを基本骨格とし、片方のFabをCD3εに対する結合ドメインに置き換えた形の分子を作製した。この際、基本骨格とするIgGのFcとしては、上述した場合と同様に、FcgR(Fcγ受容体)への結合性が減弱されたサイレント型Fcを用いた。EGFRに対する結合ドメインとして、Cetuximabの可変領域であるCetuximab-VH(配列番号:15)、Cetuximab-VL(配列番号:16)を使用した。抗体H鎖定常領域として、IgG1のC末端のGly及びLysを除去したG1d、G1dにD356K及びH435Rの変異を導入したA5、並びにG1dにK439Eの変異を導入したB3を使用し、それぞれCetuximab-VHと組み合わせたCetuximab-VH-G1d(配列番号:17)、Cetuximab-VH-A5(配列番号:18)、Cetuximab-VH-B3(配列番号:19)を、参考実施例1の方法にしたがって調製した。なお、抗体H鎖定常領域の名称をH1とした場合、可変領域にCetuximab-VHを持つ抗体のH鎖に対応する配列はCetuximab-VH-H1のように示した。
ここで、アミノ酸の改変を示す場合には、D356Kのように示した。最初のアルファベット(D356KのDに該当)は、改変前のアミノ酸残基を一文字表記で示した場合のアルファベットを意味し、それに続く数字(D356Kの356に該当)はその改変箇所のEUナンバリングを意味し、最後のアルファベット(D356KのKに該当)は改変後のアミノ酸残基を一文字表記で示した場合のアルファベットを意味する。 (2-3) Preparation of EGFR_ERY22_CE115 Next, a molecule was prepared in which IgG for cancer antigen (EGFR) was used as a basic skeleton and one Fab was replaced with a binding domain for CD3ε. At this time, as the Fc of IgG as a basic skeleton, silent Fc with reduced binding to FcgR (Fcγ receptor) was used as in the case described above. Cetuximab-VH (SEQ ID NO: 15) and Cetuximab-VL (SEQ ID NO: 16), which are variable regions of Cetuximab, were used as binding domains for EGFR. G1d with Gly and Lys removed from the C-terminus of IgG1, A5 with D356K and H435R mutations introduced into G1d, and B3 with K439E mutation introduced into G1d as the antibody H chain constant region, and Cetuximab-VH Cetuximab-VH-G1d (SEQ ID NO: 17), Cetuximab-VH-A5 (SEQ ID NO: 18), and Cetuximab-VH-B3 (SEQ ID NO: 19) were prepared according to the method of Reference Example 1. . When the name of the antibody H chain constant region is H1, the sequence corresponding to the H chain of the antibody having Cetuximab-VH in the variable region is shown as Cetuximab-VH-H1.
Here, when an amino acid modification is indicated, it is indicated as D356K. The first alphabet (corresponding to D in D356K) means the alphabet when the amino acid residue before modification is shown in single letter notation, and the following number (corresponding to 356 in D356K) means the EU numbering of the modified part The last alphabet (corresponding to K in D356K) means the alphabet in the case where the amino acid residue after modification is indicated by a single letter.
・目的分子:EGFR_ERY22_CE115
・発現ベクターに挿入されたポリヌクレオチドによりコードされるポリペプチド:EGFR _ERY22_Hk、EGFR _ERY22_L、CE115_ERY22_Hh、CE115_ERY22_L The following combinations of expression vectors were introduced into FreeStyle293-F cells to transiently express each target molecule.
・ Target molecule: EGFR_ERY22_CE115
-Polypeptides encoded by polynucleotides inserted into expression vectors: EGFR_ERY22_Hk, EGFR_ERY22_L, CE115_ERY22_Hh, CE115_ERY22_L
得られた培養上清がAnti FLAG M2カラム(Sigma社)に添加され、当該カラムの洗浄の後、0.1 mg/mL FLAGペプチド(Sigma社)による溶出が実施された。目的分子を含む画分がHisTrap HPカラム(GE Healthcare社)に添加され、当該カラムの洗浄の後、イミダゾールの濃度勾配による溶出が実施された。目的分子を含む画分が限外ろ過によって濃縮された後、当該画分がSuperdex 200カラム(GE Healthcare社)に添加され、溶出液の単量体画分のみを回収することにより精製された各目的分子が得られた。 (2-4) Purification of EGFR_ERY22_CE115 The obtained culture supernatant was added to an Anti FLAG M2 column (Sigma), and the column was washed and then eluted with 0.1 mg / mL FLAG peptide (Sigma). . The fraction containing the target molecule was added to a HisTrap HP column (GE Healthcare), and the column was washed and then eluted with an imidazole concentration gradient. After the fraction containing the target molecule was concentrated by ultrafiltration, the fraction was added to a
(2-5-1)ヒト末梢血単核球(PBMC:Peripheral Blood Mononuclear Cell)溶液の調製
1,000単位/mLのヘパリン溶液(ノボ・ヘパリン注5千単位,ノボ・ノルディスク社)をあらかじめ100μL注入した注射器を用い、健常人ボランティア(成人)より末梢血50 mLが採取された。PBS(-)で2倍希釈した後に4等分された末梢血が、15 mLのFicoll-Paque PLUSをあらかじめ注入して遠心操作が行なわれたLeucosepリンパ球分離管(Cat. No. 227290、Greiner bio-one社)に加えられた。当該分離管の遠心分離(2,150 rpm、10分間、室温)の後、単核球画分層が分取された。10%FBSを含むDulbecco's Modified Eagle's Medium(SIGMA社、以下10%FBS/D-MEM)で単核球画分の細胞を1回洗浄した後、当該細胞を、その細胞密度が4×106 /mLになるよう、10%FBS/D-MEMを用いて調製した。このように調製した細胞溶液を、ヒトPBMC溶液として以後の試験に用いた。 (2-5) Measurement of cytotoxic activity using human peripheral blood mononuclear cells (2-5-1) Preparation of human peripheral blood mononuclear cell (PBMC) solution 1,000 units /
細胞傷害活性は、xCELLigenceリアルタイムセルアナライザー(ロシュ・ダイアグノスティックス社)を用いた細胞増殖抑制率で評価した。標的細胞には、SK-HEP-1細胞株にヒトEGFRを強制発現させて樹立したSK-pca13a細胞株を用いた。SK-pca13aをディッシュから剥離し、1×104 cells/wellとなるようにE-Plate 96プレート(ロシュ・ダイアグノスティックス社)に100μL/wellで播き、xCELLigenceリアルタイムセルアナライザーを用いて生細胞の測定を開始した。翌日xCELLigenceリアルタイムセルアナライザーからプレートを取り出し、当該プレートに各濃度(0.004、0.04、0.4、4 nM)に調製した各抗体50μLを添加した。室温にて15分間反応させた後に(2-5-1)で調製したヒトPBMC溶液50μL(2×105 cells/well)を加え、xCELLigenceリアルタイムセルアナライザーに当該プレートを再セットすることによって、生細胞の測定を開始した。反応は、5%炭酸ガス、37℃条件下にて行い、ヒトPBMC添加72時間後のCell Index値から、下式により細胞増殖抑制率(%)を求めた。なお、計算に用いたCell Index値には、抗体添加直前のCell Index値が1となるようにノーマライズした後の数値を用いた。
細胞増殖抑制率(%)=(A-B)×100/(A-1)
Aは抗体を添加していないウェルにおけるCell Index値の平均値(標的細胞とヒトPBMCのみ)、Bは各ウェルにおけるCell Index値の平均値を示す。試験はtriplicateにて行なった。 (2-5-2) Measurement of cytotoxic activity Cytotoxic activity was evaluated based on the cell growth inhibition rate using an xCELLigence real-time cell analyzer (Roche Diagnostics). As the target cell, the SK-pca13a cell line established by forcibly expressing human EGFR in the SK-HEP-1 cell line was used. SK-pca13a is detached from the dish, seeded at 100 μL / well on an E-Plate 96 plate (Roche Diagnostics) at 1 × 10 4 cells / well, and live cells using xCELLigence real-time cell analyzer Measurement was started. On the next day, the plate was taken out from the xCELLigence real-time cell analyzer, and 50 μL of each antibody prepared at each concentration (0.004, 0.04, 0.4, 4 nM) was added to the plate. After reacting at room temperature for 15 minutes, add 50 μL (2 × 10 5 cells / well) of the human PBMC solution prepared in (2-5-1) and reset the plate in the xCELLigence real-time cell analyzer. Cell measurement was started. The reaction was carried out under conditions of 5% carbon dioxide gas and 37 ° C., and the cell growth inhibition rate (%) was determined from the
Cell growth inhibition rate (%) = (AB) x 100 / (A-1)
A shows the average Cell Index value in the wells to which no antibody is added (only target cells and human PBMC), and B shows the average Cell Index value in each well. The test was performed in triplicate.
図1~6に示したように、Dual binding Fabとは、可変(Fab)領域でCD3(第1の抗原)と目的の抗原(第2の抗原)に結合するが、CD3(第1の抗原)及び目的の抗原(第2の抗原)に同時には結合しない分子である。第2の抗原と結合するためにCD3(第1の抗原)に結合する抗体のFab領域にアミノ酸改変を導入する場合、通常二つのH鎖もしくはL鎖の両方にアミノ酸改変が導入される。しかし、両方のH鎖もしくはL鎖に改変が導入されると、抗体の二つのFabがそれぞれ、二つの抗原に結合することにより、二つのFabとCD3(第1の抗原)及び目的の抗原(第2の抗原)に同時に結合し、架橋する可能性がある。したがって、抗体の一つのFabは、第3の抗原に結合する、あるいは何にも結合しないFabとし、もう一つのFabをdual binding Fabとし、CD3(第1の抗原)及び目的の抗原(第2の抗原)の架橋反応は起こらないようにする。 [Example 3] Preparation of antibody that binds to CD3 and human integrin αvβ3 but does not bind simultaneously As shown in FIGS. 1 to 6, Dual binding Fab is a variable (Fab) region of CD3 (first antigen). ) And the target antigen (second antigen), but does not bind to CD3 (first antigen) and target antigen (second antigen) at the same time. When an amino acid modification is introduced into the Fab region of an antibody that binds to CD3 (first antigen) in order to bind to the second antigen, the amino acid modification is usually introduced into both the two H chains or L chains. However, when a modification is introduced into both H chains or L chains, the two Fabs of the antibody bind to the two antigens, respectively, so that the two Fabs and CD3 (first antigen) and the target antigen ( (Second antigen) may be simultaneously bound and cross-linked. Therefore, one Fab of the antibody is a Fab that binds to the third antigen or binds nothing, and the other Fab is a dual binding Fab, and CD3 (the first antigen) and the target antigen (the second antigen). The cross-linking reaction of the antigens of
接着分子として知られているインテグリンαvβ3は、多くのがん細胞および腫瘍の周りの血管に発現していることから、腫瘍をターゲッティングする標的分子として有用であるが、一方でさまざまな正常細胞にも発現していることが知られている(Thromb Haemost. 1998 Nov;80(5):726-34.)。したがって、CD3とインテグリンαvβ3が同時に結合してしまうと正常細胞がT細胞による強力な細胞傷害活性によって傷害されてしまう可能性が考えられた。そこでCD3とインテグリンαvβ3が同時には結合しない分子が作製できれば、正常細胞に傷害を与えることなく、インテグリンαvβ3を発現する腫瘍細胞に抗EGFR抗体分子をターゲットすることができると考えられた。すなわち、片側の可変領域(Fab)でEGFRに結合し、別の可変領域で、第1の抗原であるCD3に結合、さらに第2の抗原であるインテグリンαvβ3に結合し、且つ、CD3及びインテグリンαvβ3に同時には結合しないdual binding Fab分子の取得を検討した。 (3-1) Production of an antibody that binds to CD3 and human integrin αvβ3 but does not bind at the same time Integrin αvβ3, known as an adhesion molecule, is expressed in many cancer cells and blood vessels around the tumor From the above, it is known that it is useful as a target molecule for targeting tumors, but is also expressed in various normal cells (Thromb Haemost. 1998 Nov; 80 (5): 726-34.). Therefore, it was considered that if CD3 and integrin αvβ3 are bound simultaneously, normal cells may be damaged by the strong cytotoxic activity of T cells. Therefore, if a molecule that does not bind CD3 and integrin αvβ3 at the same time could be prepared, it was thought that anti-EGFR antibody molecules could be targeted to tumor cells expressing integrin αvβ3 without damaging normal cells. That is, it binds to EGFR at one variable region (Fab), binds to CD3 as the first antigen, binds to integrin αvβ3 as the second antigen, and binds to CD3 and integrin αvβ3. We studied the acquisition of dual binding Fab molecules that do not bind simultaneously.
Dual binding Fab分子を取得する方法として、ライブラリーを利用する方法と、タンパク質に対して結合活性を有することが知られているペプチドを挿入する方法が考えられた。インテグリンαvβ3に対して結合活性を有するペプチドとして、RGD(Arg-Gly-Asp)ペプチドが知られている。そこで、片側のFabをEGFR結合ドメインとし、もう片側のFabをCD3結合ドメイン及びインテグリンαvβ3結合ドメインとするヘテロ二量化抗体であって、CD3εに結合する抗体であるCE115(重鎖可変領域 配列番号:13、軽鎖可変領域 配列番号:14)の重鎖のループ部分にRGDペプチドを挿入したヘテロ二量化抗体を参考実施例1に従って作製した。すなわち、EGFR ERY22_Hk(配列番号:20)、EGFR ERY22_L(配列番号:21)及びCE115_ERY22_L(配列番号:23)をそれぞれコードするポリヌクレオチドと共に、以下のいずれかをコードするポリヌクレオチドが挿入された一連の発現ベクターを作製した:
・CE115_2 ERY22_Hh(配列番号:24、Kabatナンバリング52b-53をそれぞれK及びNに置換)、
・CE115_4 ERY22_Hh(配列番号:25、Kabatナンバリング52b-54をそれぞれS及びNに置換)、
・CE115_9 ERY22_Hh(配列番号:26、Kabatナンバリング52a-52bの間にRGDを挿入)、
・CE115_10 ERY22_Hh(配列番号:27、Kabatナンバリング52b-52cの間にRGDを挿入)、
・CE115_12 ERY22_Hh(配列番号:28、Kabatナンバリング72-73の間にRGDを挿入)、
・CE115_17 ERY22_Hh(配列番号:29、Kabatナンバリング52b-52cをそれぞれK及びSに置換)、
・CE115_47 ERY22_Hh(配列番号:30、Kabatナンバリング98-99の間にRGDを挿入)、
・CE115_48 ERY22_Hh(配列番号:31、Kabatナンバリング99-100の間にRGDを挿入)、
・CE115_49 ERY22_Hh(配列番号:32、Kabatナンバリング100-100aの間にRGDに挿入)。
また、対照として、J. Biotech, 155, 193-202, 2011に報告されている抗体のCH3領域にRGD(Arg-Gly-Asp)ペプチドが挿入された抗体(EH240-Kn125 /EH240-Hl076/L73;配列番号33/34/35)を参考実施例1に従って作製した。CH3領域を介してインテグリンαvβ3と結合するこの分子は、CD3とインテグリンαvβ3に同時に結合することが出来ると考えられる。 (3-2) Acquisition of an antibody having a Fab region that binds to integrin αvβ3 As a method of acquiring a dual binding Fab molecule, a method using a library and a peptide known to have a binding activity to a protein The method of inserting was considered. An RGD (Arg-Gly-Asp) peptide is known as a peptide having binding activity for integrin αvβ3. Therefore, CE115 (heavy chain variable region SEQ ID NO :), which is a heterodimerized antibody in which one side of the Fab is an EGFR binding domain and the other side of the Fab is a CD3 binding domain and an integrin αvβ3 binding domain and binds to CD3ε. 13. Light chain variable region SEQ ID NO: 14) A heterodimerized antibody in which an RGD peptide was inserted into the heavy chain loop was prepared according to Reference Example 1. That is, a series of polynucleotides encoding any of the following together with polynucleotides encoding EGFR ERY22_Hk (SEQ ID NO: 20), EGFR ERY22_L (SEQ ID NO: 21) and CE115_ERY22_L (SEQ ID NO: 23), respectively. An expression vector was created:
CE115_2 ERY22_Hh (SEQ ID NO: 24, Kabat numbering 52b-53 replaced with K and N, respectively),
CE115_4 ERY22_Hh (SEQ ID NO: 25, Kabat numbering 52b-54 replaced with S and N, respectively),
CE115_9 ERY22_Hh (SEQ ID NO: 26, RGD inserted between Kabat numbering 52a-52b),
CE115_10 ERY22_Hh (SEQ ID NO: 27, RGD inserted between Kabat numbering 52b-52c),
CE115_12 ERY22_Hh (SEQ ID NO: 28, RGD inserted between Kabat numbering 72-73),
CE115_17 ERY22_Hh (SEQ ID NO: 29, replacing Kabat numbering 52b-52c with K and S, respectively),
CE115_47 ERY22_Hh (SEQ ID NO: 30, RGD inserted between Kabat numbering 98-99),
CE115_48 ERY22_Hh (SEQ ID NO: 31, insert RGD between Kabat numbering 99-100),
CE115_49 ERY22_Hh (SEQ ID NO: 32, inserted into RGD between Kabat numbering 100-100a).
As a control, an antibody (EH240-Kn125 / EH240-Hl076 / L73) in which an RGD (Arg-Gly-Asp) peptide is inserted into the CH3 region of an antibody reported in J. Biotech, 155, 193-202, 2011 ; SEQ ID NO: 33/34/35) was prepared according to Reference Example 1. This molecule that binds to integrin αvβ3 through the CH3 region is thought to be able to bind to CD3 and integrin αvβ3 simultaneously.
Fab領域にRGD(Arg-Gly-Asp)ペプチドが挿入された分子がインテグリンαvβ3と結合するかどうか、電気化学発光法(ECL法)で判定した。具体的には、0.1%BSAおよび0.1g/L塩化カルシウムおよび0.1g/L塩化マグネシウムを含むTBS溶液(希釈(+) 溶液と表記する)で希釈したbiotin-anti human IgG Ab (Southern biotech)と、5 μg/mLもしくは1 μg/mLに調製された抗体溶液と、sulfo-tagを付加したインテグリンαvβ3(R&D Systems)を、Nunc-Immuno(tm) MicroWell(tm) 96 well round plates(Nunc)の各ウェルに25 μLずつ添加し、混合した後、4℃で一晩インキュベートし、抗体抗原複合体を形成させた。0.5%BSAおよび0.1g/L塩化カルシウムおよび0.1g/L塩化マグネシウムを含むTBS溶液(ブロッキング(+) 溶液と表記する)をstreptavidin plate(MSD)各ウェルに150μLずつ加えて4℃で一晩インキュベートした。ブロッキング溶液を除いた後、0.1g/L塩化カルシウムおよび0.1g/L塩化マグネシウムを含むTBS溶液(TBS(+)溶液と表記する)250μLで3回洗浄した。抗体抗原複合体溶液を75μLずつ各ウェルに添加し、室温2時間インキュベートして、biotin-anti human IgG Abをstreptavidin plateに結合させた。抗体抗原複合体溶液を除いた後、TBS(+)溶液で3回洗浄し、READ buffer(MSD)を各ウェルに150μLずつ加え、Sector Imager 2400(MSD)でsulfo-tagの発光シグナルを検出した。 (3-3) Confirmation of binding of integrin αvβ3 and antibody Whether or not a molecule having an RGD (Arg-Gly-Asp) peptide inserted in the Fab region binds to integrin αvβ3 was determined by electrochemiluminescence method (ECL method). Specifically, biotin-anti human IgG Ab (Southern biotech) diluted with a TBS solution containing 0.1% BSA and 0.1 g / L calcium chloride and 0.1 g / L magnesium chloride (denoted as a diluted (+) solution) , Antibody solution prepared to 5 μg / mL or 1 μg / mL and integrin αvβ3 (R & D Systems) with sulfo-tag added to Nunc-Immuno (tm) MicroWell (tm) 96 well round plates (Nunc) 25 μL each was added to each well, mixed, and then incubated overnight at 4 ° C. to form antibody-antigen complexes. Add 150 μL of TBS solution (referred to as blocking (+) solution) containing 0.5% BSA, 0.1 g / L calcium chloride and 0.1 g / L magnesium chloride to each well of streptavidin plate (MSD) and incubate overnight at 4 ° C. did. After removing the blocking solution, the plate was washed 3 times with 250 μL of a TBS solution (denoted as TBS (+) solution) containing 0.1 g / L calcium chloride and 0.1 g / L magnesium chloride. 75 μL of the antibody-antigen complex solution was added to each well and incubated at room temperature for 2 hours to bind biotin-anti human IgG Ab to the streptavidin plate. After removing the antibody-antigen complex solution, the plate was washed 3 times with TBS (+) solution, 150 μL of READ buffer (MSD) was added to each well, and the sulfo-tag luminescence signal was detected with Sector Imager 2400 (MSD). .
次に、前項で作製したインテグリンαvβ3とFab領域で結合する抗体が、CD3に対する結合活性を保持しているかECL法で判定した。具体的には、0.1%BSAを含むTBS溶液(希釈(-) 溶液と表記する)で希釈したbiotin-anti human IgG Ab (Southern biotech)と、5 μg/mLもしくは1 μg/mLに調製された抗体溶液と、sulfo-tagを付加したCD3εホモ二量体タンパク質を、Nunc-Immuno(tm) MicroWell(tm) 96 well round plates(Nunc)の各ウェルに25 μLずつ添加し、混合した後、4℃で一晩インキュベートし、抗体抗原複合体を形成させた。0.5%BSAを含むTBS溶液(ブロッキング(-) 溶液と表記する)をstreptavidin plate(MSD)各ウェルに150μLずつ加えて4℃で一晩インキュベートした。ブロッキング溶液を除いた後、TBS溶液 (-)溶液250μLで3回洗浄した。抗体抗原複合体溶液を75μLずつ各ウェルに添加し、室温2時間インキュベートして、biotin-anti human IgG Abをstreptavidin plateに結合させた。抗体抗原複合体溶液を除いた後、TBS(-)溶液で3回洗浄し、READ buffer(MSD)を各ウェルに150μLずつ加え、Sector Imager 2400(MSD)でsulfo-tagの発光シグナルを検出した。 (3-4) Confirmation of binding between CD3 (CD3ε) and antibody Next, it was determined by the ECL method whether the antibody that binds to the integrin αvβ3 prepared in the previous section and the Fab region has binding activity to CD3. Specifically, biotin-anti human IgG Ab (Southern biotech) diluted with TBS solution containing 0.1% BSA (referred to as “diluted (−) solution”) was prepared to 5 μg / mL or 1 μg / mL. Add 25 μL of the antibody solution and CD3ε homodimeric protein with sulfo-tag to each well of Nunc-Immuno (tm) MicroWell (tm) 96 well round plates (Nunc), mix, Incubated overnight at 0 ° C. to form antibody-antigen complexes. 150 μL of a TBS solution containing 0.5% BSA (referred to as blocking (−) solution) was added to each well of the streptavidin plate (MSD) and incubated overnight at 4 ° C. After removing the blocking solution, it was washed 3 times with 250 μL of TBS solution (−) solution. 75 μL of the antibody-antigen complex solution was added to each well and incubated at room temperature for 2 hours to bind biotin-anti human IgG Ab to the streptavidin plate. After removing the antibody-antigen complex solution, it was washed 3 times with TBS (-) solution, 150 μL of READ buffer (MSD) was added to each well, and the luminescence signal of sulfo-tag was detected with Sector Imager 2400 (MSD). .
前項までの結果から、インテグリンαvβ3に対して結合活性を有し、かつ、CD3に対して結合活性を有する分子が得られた。次に、前項までに作製されたFab領域がCD3(CD3ε)及びインテグリンαvβ3と同時に結合するか判定した。 (3-5) Confirming that integrin αvβ3 and CD3 do not bind to the Fab region at the same time by the ECL method From the results up to the previous section, molecules having binding activity to integrin αvβ3 and binding activity to CD3 was gotten. Next, it was determined whether or not the Fab region prepared up to the previous section binds simultaneously with CD3 (CD3ε) and integrin αvβ3.
以上の結果から、一つのFabでCD3(CD3ε)、インテグリンαvβ3にそれぞれ結合し、かつ、CD3(CD3ε)及びインテグリンαvβ3に同時には結合しないdual binding Fab分子の特性を有する抗体を創製することができた。本実施例では、第1の抗原であるCD3に結合する可変領域を有する抗体に対して、当該可変領域に第2の抗原であるインテグリンαvβ3に結合するRGDペプチドをFabに挿入することで、第2の抗原に対する結合活性を付与し、且つ、CD3と第2の抗原に同時には結合しない分子を取得することが出来た。同様の方法で、WO2006036834に例示されているようなタンパク質に対して結合活性を有するペプチドをFab中のループに挿入することで、任意の第2の抗原に対して結合活性を有するdual binding Fab分子を取得することができる。その他、タンパク質に対して結合活性を示すペプチドは、当業者に公知の方法を用いてペプチドライブラリを作製し、所望の活性を有するペプチドを選択することで取得することができる(Pasqualini R., Nature, 1996, 380 (6572) :364-6)。さらに、実施例5で記載したようなFab中のループを長く改変した(延長した)抗原結合分子のライブラリーを用いることで、任意の第2の抗原に対して結合活性を有するdual binding Fab分子を創製することが可能であると考えられる。第1の抗原に対する可変領域は、当業者公知の様々な方法で取得することが可能であることから、このようなライブラリーを用いることで、任意の第1の抗原と任意の第2の抗原に対して結合活性を有し、かつ該第1の抗原及び該第2の抗原に同時には結合することができないdual binding Fab分子を創製することが可能であると言える。 (3-6) Consideration that integrin αvβ3 and CD3 do not bind to Fab region simultaneously by ECL method From the above results, one Fab binds to CD3 (CD3ε) and integrin αvβ3 respectively, and CD3 (CD3ε) and An antibody having the characteristics of a dual binding Fab molecule that does not bind to integrin αvβ3 simultaneously can be created. In this example, for an antibody having a variable region that binds to CD3, which is the first antigen, an RGD peptide that binds to integrin αvβ3, which is the second antigen, is inserted into the Fab. It was possible to obtain a molecule that imparts binding activity to the two antigens and does not bind to CD3 and the second antigen at the same time. In the same manner, a dual binding Fab molecule that has binding activity to any second antigen by inserting a peptide having binding activity to a protein as exemplified in WO2006036834 into a loop in the Fab. Can be obtained. In addition, a peptide showing binding activity to a protein can be obtained by preparing a peptide library using a method known to those skilled in the art and selecting a peptide having a desired activity (Pasqualini R., Nature , 1996, 380 (6572): 364-6). Furthermore, by using a library of antigen-binding molecules in which the loop in the Fab has been modified (extended) as described in Example 5, a dual binding Fab molecule having binding activity to any second antigen Is considered possible. Since the variable region for the first antigen can be obtained by various methods known to those skilled in the art, by using such a library, any first antigen and any second antigen can be obtained. It can be said that it is possible to create a dual binding Fab molecule that has binding activity to and that cannot simultaneously bind to the first antigen and the second antigen.
(4-1)CD3及びヒトTLR2に結合するが、同時には結合しない抗体の作製
パターン認識受容体として知られているTLR2は、主にマクロファージ、樹状細胞やB細胞などの免疫細胞に発現しており、免疫細胞を活性化する標的分子として有用である。また、TLR2は、上皮細胞や内皮細胞など免疫細胞以外の正常細胞にも発現していることが知られている。がん抗原とCD3を同時に結合することにより、腫瘍環境中にCD3を発現するT細胞がリクルートされ、T細胞によってがん細胞が傷害されるが、同時にがん抗原とTLR2とが同時に結合することにより、腫瘍環境中にTLR2を発現する免疫細胞もリクルートし、活性化できることが考えられた。T細胞によって傷害されたがん細胞を、TLR2によってリクルートされた免疫細胞が取り込み、抗原をプロセシングし、HLAに提示することによりT細胞を活性化することができるため、T細胞をより強力に活性化できるとともに、獲得免疫も誘導できる可能性がある。しかし、CD3とTLR2が同時に結合してしまうと免疫細胞及び正常細胞がT細胞による強力な細胞傷害活性によって傷害されてしまう可能性が考えられた。そこでCD3とTLR2が同時には結合しない分子を作製できれば、TLR2を発現する免疫細胞及び正常細胞に傷害を与えることなく、これらの細胞をリクルートできると考えられた。すなわち、片側の可変領域(Fab)でEGFRに結合し、別の可変領域で、第1の抗原であるCD3に結合し、さらに第2の抗原であるTLR2に結合し、且つ、CD3及びTLR2に同時には結合しないdual binding Fab分子の取得を検討した。 [Example 4] Preparation of antibody that binds to CD3 and human toll-like receptor 2 (TLR2) but does not bind simultaneously (4-1) Preparation pattern of antibody that binds to CD3 and human TLR2 but does not bind simultaneously TLR2, which is known as a recognition receptor, is mainly expressed in immune cells such as macrophages, dendritic cells and B cells and is useful as a target molecule for activating immune cells. In addition, TLR2 is known to be expressed in normal cells other than immune cells such as epithelial cells and endothelial cells. By simultaneously binding the cancer antigen and CD3, the T cells that express CD3 are recruited into the tumor environment, and the cancer cells are damaged by the T cell. At the same time, the cancer antigen and TLR2 are simultaneously bound. This suggested that immune cells that express TLR2 in the tumor environment can also be recruited and activated. Cancer cells injured by T cells can be activated by immune cells recruited by TLR2 and activated by processing antigens and presenting them to HLA, making T cells more active And may also induce acquired immunity. However, when CD3 and TLR2 were simultaneously bound, it was considered that immune cells and normal cells might be damaged by the strong cytotoxic activity of T cells. Therefore, if a molecule that does not bind CD3 and TLR2 at the same time can be produced, it was considered that these cells could be recruited without damaging immune cells and normal cells that express TLR2. That is, it binds to EGFR in one variable region (Fab), binds to CD3 as the first antigen in another variable region, further binds to TLR2 as the second antigen, and binds to CD3 and TLR2. The acquisition of dual binding Fab molecules that do not bind simultaneously was investigated.
ヒトTLR2に対して結合活性を有するペプチドとして、RWGYHLRDRKYKGVRSHKGVPRペプチド(配列番号:36)が知られている。そこで、片側のFabをEGFR結合ドメインとし、もう片側のFabをCD3結合ドメイン及びTLR2結合ドメインとするヘテロ二量化抗体であって、CD3εに結合する抗体であるCE115(重鎖可変領域 配列番号:13、軽鎖可変領域 配列番号:14)の重鎖のループ部分にTRL2結合ペプチドを挿入したヘテロ二量化抗体抗体を参考実施例1に従って作製した。すなわち、EGFR ERY22_Hk(配列番号:20)、EGFR ERY22_L(配列番号:21)、及びCE115_ERY22_L(配列番号:23)をそれぞれコードするポリヌクレオチドと共に、以下のいずれかをコードするポリヌクレオチドが挿入された一連の発現ベクターを作製した:
・CE115_DU21 ERY22_Hh(配列番号:37、Kabatナンバリング52b-52cの間にTRL2結合ペプチドを挿入)、
・CE115_DU22 ERY22_Hh(配列番号:38、Kabatナンバリング52b-52cの間にTRL2結合ペプチドを挿入)、
・CE115_DU26 ERY22_Hh(配列番号:39、Kabatナンバリング72-73の間にTRL2結合ペプチドを挿入)、
・CE115_DU27 ERY22_Hh(配列番号:40、Kabatナンバリング72-73の間にTRL2結合ペプチド挿入)。
また、対照として、TLR2結合ペプチドをCH3領域C末端に付加した抗体(CE115_ ERY22_DU42_Hh, 配列番号:41)、及びTLR2結合ペプチド両端にCys残基を持つペプチドをCH3領域C末端に付加した抗体(CE115_ ERY22_DU43_Hh, 配列番号:42)を参考実施例1に従って作製した。CH3領域を介してTLR2と結合するこの分子は、CD3とTLR2に同時に結合することが出来ると考えられる。 (4-2) Acquisition of antibody having Fab region that binds to TLR2 As a peptide having binding activity to human TLR2, RWGYHLRDRKYKGVRSHKGVPR peptide (SEQ ID NO: 36) is known. Therefore, CE115 (heavy chain variable region SEQ ID NO: 13), which is a heterodimerized antibody in which one Fab is an EGFR binding domain and the other Fab is a CD3 binding domain and a TLR2 binding domain, is an antibody that binds to CD3ε. , Light chain variable region SEQ ID NO: 14) A heterodimerized antibody antibody in which a TRL2-binding peptide was inserted into the heavy chain loop was prepared according to Reference Example 1. That is, a series of polynucleotides encoding any of the following together with polynucleotides encoding EGFR ERY22_Hk (SEQ ID NO: 20), EGFR ERY22_L (SEQ ID NO: 21), and CE115_ERY22_L (SEQ ID NO: 23), respectively. The following expression vectors were made:
CE115_DU21 ERY22_Hh (SEQ ID NO: 37, TRL2-binding peptide inserted between Kabat numbering 52b-52c),
CE115_DU22 ERY22_Hh (SEQ ID NO: 38, TRL2-binding peptide inserted between Kabat numbering 52b-52c),
CE115_DU26 ERY22_Hh (SEQ ID NO: 39, TRL2-binding peptide inserted between Kabat numbering 72-73),
CE115_DU27 ERY22_Hh (SEQ ID NO: 40, TRL2-binding peptide inserted between Kabat numbering 72-73).
In addition, as a control, an antibody (CE115_ERY22_DU42_Hh, SEQ ID NO: 41) having a TLR2-binding peptide added to the C-terminus of the CH3 region, and an antibody (CE115_ ERY22_DU43_Hh, SEQ ID NO: 42) was prepared according to Reference Example 1. This molecule that binds to TLR2 via the CH3 region is thought to be able to bind to CD3 and TLR2 simultaneously.
Fab領域にTLR2結合ペプチドが挿入された分子がTLR2と結合するかどうか、電気化学発光法(ECL法)で判定した。具体的には、0.1%BSAを含むTBS溶液(希釈(-) 溶液と表記する)で希釈したbiotin-anti human IgG Ab (Southern biotech)と、5 μg/mLもしくは1 μg/mLに調製された抗体溶液と、sulfo-tagを付加したTLR2(abnova)を、Nunc-Immuno(tm) MicroWell(tm) 96 well round plates(Nunc)の各ウェルに25 μLずつ添加し、混合した後、4℃で一晩インキュベートし、抗体抗原複合体を形成させた。0.5%BSAを含むTBS溶液(ブロッキング(-) 溶液と表記する)をstreptavidin plate(MSD)各ウェルに150μLずつ加えて4℃で一晩インキュベートした。ブロッキング溶液を除いた後、TBS(-)溶液250μLで3回洗浄した。抗体抗原複合体溶液を75μLずつ各ウェルに添加し、室温2時間インキュベートして、biotin-anti human IgG Abをstreptavidin plateに結合させた。抗体抗原複合体溶液を除いた後、TBS(-)溶液で3回洗浄し、READ buffer(MSD)を各ウェルに150μLずつ加え、Sector Imager 2400(MSD)でsulfo-tagの発光シグナルを検出した。 (4-3) Confirmation of binding between TLR2 and antibody It was determined by electrochemiluminescence method (ECL method) whether or not a molecule having a TLR2 binding peptide inserted into the Fab region binds to TLR2. Specifically, biotin-anti human IgG Ab (Southern biotech) diluted with TBS solution containing 0.1% BSA (referred to as “diluted (−) solution”) was prepared to 5 μg / mL or 1 μg / mL. Add 25 μL of antibody solution and sulfo-tag added TLR2 (abnova) to each well of Nunc-Immuno (tm) MicroWell (tm) 96 well round plates (Nunc) and mix at 4 ° C. Incubate overnight to allow antibody-antigen complexes to form. 150 μL of a TBS solution containing 0.5% BSA (referred to as blocking (−) solution) was added to each well of the streptavidin plate (MSD) and incubated overnight at 4 ° C. After removing the blocking solution, the plate was washed 3 times with 250 μL of TBS (−) solution. 75 μL of the antibody-antigen complex solution was added to each well and incubated at room temperature for 2 hours to bind biotin-anti human IgG Ab to the streptavidin plate. After removing the antibody-antigen complex solution, it was washed 3 times with TBS (-) solution, 150 μL of READ buffer (MSD) was added to each well, and the luminescence signal of sulfo-tag was detected with Sector Imager 2400 (MSD). .
次に、前項で作製したTLR2とFab領域で結合する抗体が、CD3(CD3ε)に対する結合活性を保持しているかECL法で判定した。具体的には、0.1%BSAを含むTBS溶液(希釈(-) 溶液と表記する)で希釈したbiotin-anti human IgG Ab (Southern biotech)と、5 μg/mLもしくは1 μg/mLに調製された抗体溶液と、sulfo-tagを付加したCD3εホモ二量体タンパク質を、Nunc-Immuno(tm) MicroWell(tm) 96 well round plates(Nunc)の各ウェルに25 μLずつ添加し、混合した後、4℃で一晩インキュベートし、抗体抗原複合体を形成させた。0.5%BSAを含むTBS溶液(ブロッキング(-) 溶液と表記する)をstreptavidin plate(MSD)各ウェルに150μLずつ加えて4℃で一晩インキュベートした。ブロッキング溶液を除いた後、TBS溶液 (-)溶液250μLで3回洗浄した。抗体抗原複合体溶液を75μLずつ各ウェルに添加し、室温2時間インキュベートして、biotin-anti human IgG Abをstreptavidin plateに結合させた。抗体抗原複合体溶液を除いた後、TBS(-)溶液で3回洗浄し、READ buffer(MSD)を各ウェルに150μLずつ加え、Sector Imager 2400(MSD)でsulfo-tagの発光シグナルを検出した。 (4-4) Confirmation of binding between CD3 ( CD3ε ) and antibody Next, it was determined by the ECL method whether the antibody that binds to TLR2 and the Fab region prepared in the previous section has binding activity to CD3 (CD3ε). Specifically, biotin-anti human IgG Ab (Southern biotech) diluted with TBS solution containing 0.1% BSA (referred to as “diluted (−) solution”) was prepared to 5 μg / mL or 1 μg / mL. Add 25 μL of the antibody solution and CD3ε homodimeric protein with sulfo-tag to each well of Nunc-Immuno (tm) MicroWell (tm) 96 well round plates (Nunc), mix, Incubated overnight at 0 ° C. to form antibody-antigen complexes. 150 μL of a TBS solution containing 0.5% BSA (referred to as blocking (−) solution) was added to each well of the streptavidin plate (MSD) and incubated overnight at 4 ° C. After removing the blocking solution, it was washed 3 times with 250 μL of TBS solution (−) solution. 75 μL of the antibody-antigen complex solution was added to each well and incubated at room temperature for 2 hours to bind biotin-anti human IgG Ab to the streptavidin plate. After removing the antibody-antigen complex solution, it was washed 3 times with TBS (-) solution, 150 μL of READ buffer (MSD) was added to each well, and the luminescence signal of sulfo-tag was detected with Sector Imager 2400 (MSD). .
前項までの結果から、TLR2に対して結合活性を有し、かつ、CD3に対して結合活性を有する分子が得られた。次に、前項までに作製されたFab領域がCD3及びTLR2と同時に結合するか判定した。 (4-5) Confirmation that TLR2 and CD3 do not bind to the Fab region at the same time by the ECL method From the results up to the previous section, a molecule having binding activity to TLR2 and binding activity to CD3 was obtained. It was. Next, it was determined whether or not the Fab region prepared up to the previous section binds simultaneously with CD3 and TLR2.
以上の結果から、一つのFabでCD3、TLR2にそれぞれ結合し、かつ、CD3及びTLR2に同時には結合しないdual binding Fab分子の特性を有する抗体を創製することができた。本実施例では、第1の抗原であるCD3に結合する可変領域を有する抗体に対して、当該可変領域に第2の抗原であるTLR2に結合するRWGYHLRDRKYKGVRSHKGVPRペプチドをFabに挿入することで、第2の抗原に対する結合活性を付与し、且つ、CD3と第2の抗原に同時には結合しない分子を取得することが出来た。同様の方法で、WO2006036834に例示されているようなタンパク質に対して結合活性を有するペプチドをFab中のループに挿入することで、任意の第2の抗原に対して結合活性を有するdual binding Fab分子を取得することができる。その他、タンパク質に対して結合活性を示すペプチドは、当業者に公知の方法を用いてペプチドライブラリを作製し、所望の活性を有するペプチドを選択することで取得することができる(Pasqualini R., Nature, 1996, 380 (6572) :364-6))。さらに、実施例5で記載したようなFab中のループを長く改変した(延長した)抗原結合分子のライブラリーを用いることで、任意の第2の抗原に対して結合活性を有するdual binding Fab分子を創製することが可能であると考えられる。第1の抗原に対する可変領域は、当業者公知の様々な方法で取得することが可能であることから、このようなライブラリーを用いることで、任意の第1の抗原と任意の第2の抗原に対して結合活性を有し、該第1の抗原及び該第2の抗原に同時には結合することができない、dual binding Fab分子を創製することが可能であると言える。 (4-6) Consideration that TLR2 and CD3 do not bind to Fab region at the same time by ECL method Based on the above results, dual binding does not bind to CD3 and TLR2 at the same time. We were able to create antibodies with the characteristics of Fab molecules. In this example, by inserting an RWGYHLRDRKYKGVRSHKGVPR peptide that binds to the second antigen TLR2 into the Fab by inserting into the Fab an antibody having a variable region that binds to the first antigen CD3. It was possible to obtain a molecule that imparts the binding activity to the other antigen and does not bind to CD3 and the second antigen at the same time. In the same manner, a dual binding Fab molecule that has binding activity to any second antigen by inserting a peptide having binding activity to a protein as exemplified in WO2006036834 into a loop in the Fab. Can be obtained. In addition, a peptide showing binding activity to a protein can be obtained by preparing a peptide library using a method known to those skilled in the art and selecting a peptide having a desired activity (Pasqualini R., Nature , 1996, 380 (6572): 364-6)). Furthermore, by using a library of antigen-binding molecules in which the loop in the Fab has been modified (extended) as described in Example 5, a dual binding Fab molecule having binding activity to any second antigen Is considered possible. Since the variable region for the first antigen can be obtained by various methods known to those skilled in the art, by using such a library, any first antigen and any second antigen can be obtained. It can be said that it is possible to create a dual binding Fab molecule that has a binding activity to the first antigen and cannot simultaneously bind to the first antigen and the second antigen.
(5-1)第2の抗原に結合しうるペプチドの挿入個所と長さの検討
片側の可変領域(Fab)でがん抗原に結合し、もう一方の可変領域で、第1の抗原であるCD3に結合、さらに第2の抗原に結合し、且つ、CD3及び第2の抗原に同時には結合しないdual binding Fab分子の取得を検討した。片側のFabをEGFR結合ドメインとし、もう片側のFabをCD3結合ドメインとするヘテロ二量化抗体であって、CD3εに結合する抗体であるCE115の重鎖のループ部分にGGSペプチドを挿入したヘテロ二量化抗体を参考実施例1に従って作製した。 [Example 5] Modification of antibody for preparation of antibody that binds to CD3 and second antigen (5-1) Examination of insertion position and length of peptide capable of binding to second antigen Variable region on one side (Fab ) Binds to the cancer antigen, binds to the first antigen CD3 in the other variable region, binds to the second antigen, and does not bind to CD3 and the second antigen simultaneously. We studied the acquisition of Fab molecules. Heterodimerized antibody with one side of Fab as EGFR binding domain and the other side of Fab as CD3 binding domain, with GGS peptide inserted into the loop part of CE115 heavy chain antibody that binds to CD3ε The antibody was produced according to Reference Example 1.
作製した各種の抗体がCD3εへの結合性を維持しているかどうかをBiacoreT100によって確認した。CM5チップにストレプトアビジンを介してビオチン化CD3εエピトープペプチドを結合させ、作製した抗体をアナライトとして流し、結合アフィニティーを解析した。 (5-2) Confirmation of binding of CE115 antibody inserted with GGS peptide to CD3ε It was confirmed by BiacoreT100 whether the various antibodies produced maintained the binding property to CD3ε. A biotinylated CD3ε epitope peptide was bound to a CM5 chip via streptavidin, the prepared antibody was run as an analyte, and the binding affinity was analyzed.
ここで挿入もしくは置換されるペプチドのアミノ酸配列を、部位特異的変異誘発法(Kunkelら(Proc. Natl. Acad. Sci. U.S.A. (1985) 82, 488-492))やOverlap extension PCR等の公知の方法に従ってランダムに改変し、上述の方法に従って各改変体の結合活性等を比較し、アミノ酸配列を改変しても目的の活性を示すことのできる挿入、置換個所やそのアミノ酸の種類、長さを決定することでライブラリーを作製することができる。 That is, it was shown that by using such peptide-inserted CE115 to obtain an antibody that binds to the second antigen, an antibody that can bind to CD3 and the second antigen but does not bind simultaneously can be produced. .
The amino acid sequence of the peptide to be inserted or substituted here is known by site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) or overlap extension PCR. Randomly modified according to the method, comparing the binding activity etc. of each variant according to the method described above, the insertion, substitution position and the type and length of the amino acid that can show the desired activity even if the amino acid sequence is modified By determining, a library can be prepared.
(6-1)CD3及び第2の抗原に結合する抗体の取得のための抗体ライブラリについて(Dual Fab Libraryとも呼ぶ)
第1の抗原としてCD3(CD3ε)を選択し、CD3(CD3ε)と任意の第2の抗原に結合する抗体を取得する方法として、以下の6つが例示される。
1.第1の抗原に結合するFabドメインに第2の抗原に結合するペプチドもしくはポリペプチドを挿入する方法(実施例3と4で示されたペプチド挿入以外にも、Angew Chem Int Ed Engl. 2013 Aug 5;52(32):8295-8に例示されるようにG-CSFを挿入する方法がある。)結合するペプチドやポリペプチドは、ペプチドもしくはポリペプチドを提示したライブラリから取得可能であるが、さらに天然に存在するタンパク質の全体もしくはその一部を利用することが可能である。
2.実施例5で示されたようにFab中のループを長く改変(延長)することができる位置に様々なアミノ酸が出現するような抗体ライブラリを作製して任意の第2の抗原に対して結合活性を有するFabを、抗体ライブラリから抗原への結合活性を指標に取得する方法
3.あらかじめCD3に対して結合することが知られているFabドメインから部位特異的変異法によって作製した抗体を用いて、CD3との結合活性を維持するアミノ酸を同定し、同定されたアミノ酸が出現するような抗体ライブラリから任意の第2の抗原に対して結合活性を有するFabを、抗体ライブラリから抗原への結合活性を指標に取得する方法
4.3の方法において、さらに、Fab中のループを長く改変(延長)することができる位置に様々なアミノ酸が出現するような抗体ライブラリを作製して任意の第2の抗原に対して結合活性を有するFabを、抗体ライブラリから抗原への結合活性を指標に取得する方法
5.1.2.3.4.の方法において、糖鎖付加配列(例えばNxS, NxT、xはP以外のアミノ酸)が出現するように改変し、糖鎖レセプターが認識する糖鎖を付加させる方法(例えばハイマンノース型糖鎖を付加し、ハイマンノースレセプターが認識する。ハイマンノース型糖鎖は抗体発現時にキフネンシンを添加することで得られることが知られている(MAbs. 2012 Jul-Aug;4(4):475-87))
6.1.2.3.4の方法において、ループ部位や各種アミノ酸へ改変することが可能であった部位にCys、Lysもしくは非天然アミノ酸を挿入または置換して、第2の抗原に結合するドメイン(ポリペプチドや糖鎖、TLRアゴニストに代表される核酸)を共有結合で付加する方法(Antibody drug conjugateに代表される方法であり、Cys、Lysもしくは非天然アミノ酸へ共有結合で結合させる方法、mAbs 6:1, 34-45; January/February 2014、WO2009/134891A2、Bioconjug Chem. 2014 Feb 19;25(2):351-61に記載されている)
上記の方法を用いて、第1の抗原と第2の抗原と結合し、互いに同時に結合しないDual binding Fabが得られ、任意の第3の抗原と結合するドメイン(もう一方の可変領域と呼び、実施例1に記載されている)とは、当業者公知の方法、例えば共通L鎖、Cross mab、Fab arm exchange法、によって組み合わせることができる。 [Example 6] Library design for obtaining antibodies that bind to CD3 and second antigen (6-1) Antibody library for obtaining antibodies that bind to CD3 and second antigen (also known as Dual Fab Library) Call)
The following six methods are exemplified as methods for selecting CD3 (CD3ε) as the first antigen and obtaining an antibody that binds to CD3 (CD3ε) and any second antigen.
1. A method of inserting a peptide or polypeptide that binds to the second antigen into the Fab domain that binds to the first antigen (in addition to the peptide insertion shown in Examples 3 and 4, Angew Chem Int Ed Engl. 2013
2. As shown in Example 5, an antibody library in which various amino acids appear at a position where a loop in a Fab can be modified (extended) long and binding activity to an arbitrary second antigen is prepared. 2. A method for obtaining a Fab having an antibody from an antibody library using an antigen binding activity as an index Using an antibody created by site-directed mutagenesis from a Fab domain that is known to bind to CD3 in advance, the amino acids that maintain the binding activity to CD3 are identified, and the identified amino acids appear. In the method 4.3 of obtaining a Fab having binding activity to an arbitrary second antigen from an antibody library using the binding activity to the antigen from the antibody library as an index, the loop in the Fab is further modified to be longer An antibody library in which various amino acids appear at a position where (extension) can be made, and Fab having binding activity to any second antigen is used as an index from the binding activity to the antigen from the antibody library. How to obtain 5.1.2.3.4. In this method, the glycosylation sequence (for example, NxS, NxT, where x is an amino acid other than P) is modified so that the sugar chain recognized by the sugar chain receptor is added (for example, a high-mannose sugar chain is added). It is known that high mannose type sugar chains can be obtained by adding kifunensine during antibody expression (MAbs. 2012 Jul-Aug; 4 (4): 475-87))
In the method of 6.1.2.3.4, Cys, Lys, or an unnatural amino acid is inserted or substituted at a loop site or a site that could be modified to various amino acids, and binds to a second antigen. A method of covalently adding a domain (polypeptide, sugar chain, nucleic acid represented by a TLR agonist) (a method represented by an antibody drug conjugate, a method of covalently binding to Cys, Lys or an unnatural amino acid, mAbs 6: 1, 34-45; January / February 2014, WO2009 / 134891A2, Bioconjug Chem. 2014
Using the above method, a dual binding Fab that binds to the first antigen and the second antigen and does not bind to each other at the same time is obtained, and a domain that binds to any third antigen (referred to as the other variable region, (Described in Example 1) can be combined by methods known to those skilled in the art, such as common L chain, Cross mab, Fab arm exchange method.
CD3(CD3ε)結合抗体のテンプレート配列としてVH領域はCE115HA000(配列番号:52)、VL領域はGLS3000(配列番号:53)が選定された。それぞれ、抗原結合に関与すると考えられる部位に参考実施例1に従ってアミノ酸改変を行った。また、H鎖の定常領域はpE22Hh(天然IgG1のCH1以降の配列にL234A, L235A, N297A, D356C, T366S, L368A, Y407Vの改変を加え、C末端のGK配列を欠失させてDYKDDDDK配列(配列番号:89)を付加した配列、配列番号:54)とし、L鎖定常領域はKappa鎖(配列番号:55)を用いた。改変を行った部位は表3に示されている。CD3(CD3ε)結合活性評価のために、1アミノ酸改変抗体はOne arm抗体(天然型のIgGのうち片方のFabドメインを欠損している抗体)として取得した。具体的には、H鎖の改変の場合は、改変されたH鎖が定常領域pE22Hhと連結されたものとKn010G3(天然型IgG1の216番目以降のアミノ酸配列にC220S, Y349C, T366W、H435Rの改変を加えたもの、配列番号:56)とkappa鎖が3'側に連結されたGLS3000を用い、L鎖の改変の場合は改変されたL鎖の3'側にKappa鎖が連結された配列と、H鎖として3'側にpE22Hhが連結されたCE115HA000とKn010G3を用いて、FreeStyle293細胞で発現・精製した(参考実施例1の方法を用いた)。 (6-2) Production of 1 amino acid modified antibody of CD3 (CD3ε) binding antibody using site-directed mutagenesis The template sequence of CD3 (CD3ε) binding antibody is V115 as CE115HA000 (SEQ ID NO: 52), VL as GLS3000 (SEQ ID NO: 53) was selected. In each case, amino acid modification was carried out according to Reference Example 1 at a site considered to be involved in antigen binding. The constant region of the H chain is pE22Hh (L234A, L235A, N297A, D356C, T366S, L368A, Y407V is added to the sequence after CH1 of natural IgG1, and the GK sequence at the C terminus is deleted to obtain the DYKDDDDK sequence (sequence No .: 89) added sequence, SEQ ID NO: 54), and the K chain chain (SEQ ID NO: 55) was used as the L chain constant region. The modified sites are shown in Table 3. For evaluation of CD3 (CD3ε) binding activity, a 1 amino acid-modified antibody was obtained as a One arm antibody (an antibody lacking one Fab domain of natural IgG). Specifically, in the case of modification of the H chain, the modified H chain is linked to the constant region pE22Hh and Kn010G3 (the amino acid sequence after the 216th amino acid sequence of natural IgG1 is modified with C220S, Y349C, T366W, H435R , SEQ ID NO: 56) and GLS3000 in which the kappa chain is linked to the 3 ′ side, and in the case of modification of the L chain, a sequence in which the Kappa chain is linked to the 3 ′ side of the modified L chain It was expressed and purified in FreeStyle293 cells using CE115HA000 and Kn010G3 in which pE22Hh was linked to the 3 ′ side as the H chain (the method of Reference Example 1 was used).
(6-2)で構築および発現精製された1アミノ酸改変体はBiacoreT200(GE Healthcare)を用いて評価された。Sensor chip CM4(GE Healthcare)上にアミノカップリング法でCD3εホモ二量体タンパク質を適当量固定化した後、アナライトとして適切な濃度の抗体をインジェクトし、センサーチップ上のCD3εホモ二量体タンパク質と相互作用させた。その後、10 mmol/L Glycine-HCl (pH1.5) をインジェクトし、センサーチップを再生した。測定は25 ℃で行い、ランニングバッファーにはHBS-EP+(GE Healthcare)を用いた。測定した結果を、結合量と測定で得られたセンサーグラムに対してsingle-cycle kinetics model (1:1binding RI=0)を使って解離定数KD (M) を算出した。各パラメーターの算出には Biacore T200 Evaluation Software (GE Healthcare)を用いた。 (6-3) Evaluation of CD3 binding of 1-amino acid-modified antibody The 1-amino acid-modified product constructed and expressed and purified in (6-2) was evaluated using Biacore T200 (GE Healthcare). After fixing an appropriate amount of CD3ε homodimer protein on Sensor chip CM4 (GE Healthcare) by amino coupling method, an appropriate concentration of antibody is injected as an analyte, and CD3ε homodimer on the sensor chip is injected. Interacted with protein. Thereafter, 10 mmol / L Glycine-HCl (pH 1.5) was injected to regenerate the sensor chip. The measurement was performed at 25 ° C., and HBS-EP + (GE Healthcare) was used as a running buffer. Based on the measurement results, the dissociation constant K D (M) was calculated using a single-cycle kinetics model (1: 1 binding RI = 0) with respect to the binding amount and the sensorgram obtained by the measurement. Biacore T200 Evaluation Software (GE Healthcare) was used for calculation of each parameter.
改変前の抗体であるCE115HA000に対して各種H鎖改変体の結合量の比の結果を表4に示す。すなわち、CE115HA000を含む抗体の結合量をX、H鎖1アミノ酸改変体の結合量をYとしたときの、Z(結合量の比)=Y/Xの値である。このとき、図18に示すようにZが0.8未満の場合には、センサーグラムから結合量が非常に少ないことが認められ、正しく解離定数KD (M)が算出できない可能性が示唆された。次に、CE115HA000に対して各種H鎖改変体の解離定数KD (M)の比(=CE115HA000のKD値/改変体のKD値)を表5に示す。
表4に示されたZが0.8以上の場合には、改変前の抗体であるCE115HA000に対して結合を維持していると考えられることから、これらのアミノ酸が出現するようにデザインされた抗体ライブラリがDual Fab Libraryとなりうる。 (6-3-1) Modification of H chain Table 4 shows the results of the ratios of the binding amounts of various H chain variants to CE115HA000, which is an antibody before modification. That is, Z (binding amount ratio) = Y / X, where X is the binding amount of the antibody containing CE115HA000 and Y is the binding amount of the
When Z shown in Table 4 is 0.8 or more, it is considered that the binding to the pre-modified antibody CE115HA000 is considered, so the antibody library designed so that these amino acids appear. Can be a Dual Fab Library.
改変前の抗体であるGLS3000に対して各種L鎖改変体の結合量の比の結果を表6に示す。すなわち、GLS3000を含む抗体の結合量をX、L鎖1アミノ酸改変体の結合量をYとしたときの、Z(結合量の比)=Y/Xの値である。このとき、図18に示すようにZが0.8未満の場合には、センサーグラムから結合量が非常に少ないことが認められ、正しく解離定数KD (M)が算出できない可能性が示唆された。次に、GLS3000に対して各種L鎖改変体の解離定数KD (M)の比を表7に示す。
表6に示されたZが0.8以上の場合には、改変前の抗体であるGLS3000に対して結合を維持していると考えられることから、これらのアミノ酸が出現するようにデザインされた抗体ライブラリがDual Fab Libraryとなりうる。 (6-3-2) Modification of L chain Table 6 shows the results of the ratios of the binding amounts of various L chain variants to the antibody GLS3000 before modification. That is, Z (binding amount ratio) = Y / X, where X is the binding amount of the antibody containing GLS3000 and Y is the binding amount of the
When Z shown in Table 6 is 0.8 or more, it is considered that the binding to GLS3000, which is an antibody before modification, is maintained, so an antibody library designed so that these amino acids appear Can be a Dual Fab Library.
ECM(Extracellular matrix;細胞外マトリックス)は、細胞外の構成成分の一つであり、生体内の様々な部位に存在している。そのため、ECMに強く結合する抗体は血中動態が悪くなる(半減期が短くなる)ことが知られている(WO2012093704A1)。そこで、抗体ライブラリで出現するアミノ酸についても、ECM結合が増強されないアミノ酸を選択することが好ましい。 (6-4) ECM (Extracellular matrix) binding evaluation of 1-amino acid-modified antibody ECM (Extracellular matrix) is one of the components of the extracellular area, and it can be found at various sites in the body. Existing. Therefore, it is known that an antibody that binds strongly to ECM has poor blood kinetics (shorter half-life) (WO2012093704A1). Therefore, it is preferable to select amino acids that do not enhance ECM binding for amino acids that appear in the antibody library.
表8(H鎖)、表9(L鎖)に示された値のうち、複数改変によるECM結合増強の効果を考慮し、10倍までを有効としてDual Fab Libraryに採用した。 For each H chain or L chain variant, an antibody was obtained by the method shown in (6-2). Next, ECM binding was evaluated according to the method of Reference Example 2. The value obtained by dividing the ECM binding value (ECL response) of each variant by the antibody ECM binding value of MRA (H chain SEQ ID NO: 57, L chain SEQ ID NO: 58) on the same plate or on the same day of execution. Are shown in Table 8 (H chain) and Table 9 (L chain). As shown in Tables 8 and 9, some modifications tended to enhance ECM binding.
Of the values shown in Table 8 (H chain) and Table 9 (L chain), the effect of enhancing ECM binding by multiple modifications was taken into consideration, and up to 10 times was effective and adopted in the Dual Fab Library.
実施例5において、各箇所においてGGS配列を用いてCD3(CD3ε)への結合を失うことなくペプチドを挿入できることが示された。Dual Fab Libraryにおいても、ループ延長が可能となれば、より多種類の分子が含まれる(多様性が大きいとも表現する)ライブラリとなり、多様な第2の抗原に結合するFabドメインの取得が可能になると考えられた。そこで、ペプチド挿入に伴って結合活性が低下することが予想されたため、CE115HA000配列にCD3εへの結合活性が高くなるようにV11L/D72A/L78I/D101Q改変を加えてpE22Hhを連結した配列に、実施例5と同様にGGSリンカーを挿入した分子を作製し、CD3結合を評価した。GGS配列は、Kabat numberingで99-100の間に挿入された。抗体分子は、One arm抗体として発現された。具体的には、GGSリンカーを含む前述のH鎖とKn010G3(配列番号:56)とL鎖としてGLS3000(配列番号:53)とKappa配列(配列番号:55)を連結した配列を採用し、参考実施例1に従って発現精製が行われた。 (6-5) Examination of peptide insertion location and length to enhance library diversity In Example 5, the peptide can be inserted without losing binding to CD3 (CD3ε) using the GGS sequence at each location. It has been shown. If the loop can be extended in the Dual Fab Library, it will be a library that includes more types of molecules (also expressed as more diversity), and it will be possible to obtain Fab domains that bind to a variety of second antigens. It was thought to be. Therefore, since the binding activity was expected to decrease as the peptide was inserted, the V11L / D72A / L78I / D101Q modification was added to the CE115HA000 sequence so that the binding activity to CD3ε was increased. In the same manner as in Example 5, a molecule into which a GGS linker was inserted was prepared, and CD3 binding was evaluated. The GGS sequence was inserted between 99-100 with Kabat numbering. The antibody molecule was expressed as a One arm antibody. Specifically, the above-mentioned H chain containing a GGS linker, Kn010G3 (SEQ ID NO: 56), and a GLS3000 (SEQ ID NO: 53) and Kappa sequence (SEQ ID NO: 55) linked as the L chain are used as a reference. Expression purification was performed according to Example 1.
GGSペプチドを挿入した改変抗体のCD3εへの結合は実施例6に記載の方法でBiacoreを用いて実施された。その結果、表10に示すようにループ部位へのGGSリンカーの挿入が可能であることが明らかになった。特に抗原結合に重要であるH鎖CDR3領域にGGSリンカーを挿入することが可能であり、3,6,9アミノ酸のいずれの挿入でもCD3εへの結合が維持された。本検討ではGGSリンカーを用いて検討したが、GGSではなく各種アミノ酸が出現する抗体ライブラリであっても良いと考えられる。 (6-6) Confirmation of binding of CE115 antibody inserted with GGS peptide to CD3 Binding of the modified antibody inserted with GGS peptide to CD3ε was performed using Biacore according to the method described in Example 6. As a result, it was revealed that the GGS linker can be inserted into the loop site as shown in Table 10. In particular, it was possible to insert a GGS linker in the H chain CDR3 region, which is important for antigen binding, and binding to CD3ε was maintained by any insertion of 3, 6, 9 amino acids. In this study, the GGS linker was used for the study, but it may be an antibody library in which various amino acids appear instead of GGS.
(6-6)ではGGSリンカーを用いて3,6,9アミノ酸の挿入が可能であり、3,6,9アミノ酸を挿入したライブラリを作成して、通常のPhage display法に代表されるような抗体取得法を用いれば第2の抗原に結合する抗体を取得することが出来ると考えられた。そこで、CDR3への挿入が6アミノ酸である場合に、NNS塩基(各種アミノ酸が出現する)を用いて、6アミノ酸を挿入する部位に様々なアミノ酸が出現してもCD3との結合を保持するか検討した。結合活性の低下が予想されたことからCE115HA000よりもCD3ε結合活性が高いCE115HA340配列(配列番号:59)のCDR3中の99-100(Kabat numbering)の間に6アミノ酸が挿入されるようにNNS塩基を用いてプライマーが設計された。抗体分子は、One arm抗体として発現された。具体的には、前述の改変を含む含む前述のH鎖とKn010G3(配列番号:56)とL鎖としてGLS3000(配列番号:53)とKappa配列(配列番号:55)を連結した配列を採用し、参考実施例1に従って発現精製が行われた。取得された改変抗体は(6-3)に記載された方法で結合が評価された。その結果を表11に示す。アミノ酸を延長した部位に各種アミノ酸が出現した場合でも、CD3(CD3ε)への結合性が保持されることが明らかになった。さらに、非特異的結合が増強されるか否かを参考実施例2に示された方法で評価した結果を表12に示す。その結果、CDR3の伸長したループ内に正電荷を側鎖に持つアミノ酸が多く含まれるとECMへの結合が増強されることから、ループ内に3個以上の正電荷を側鎖に持つアミノ酸が出現しないことが望まれた。 (6-7) Examination of library insertion into H chain CDR3 using NNS base In (6-6), 3, 6, 9 amino acids can be inserted using a GGS linker. It was considered that an antibody that binds to the second antigen can be obtained by creating an inserted library and using an antibody obtaining method represented by the ordinary Phage display method. Therefore, when the insertion into CDR3 is 6 amino acids, using NNS base (various amino acids appear), can the binding to CD3 be maintained even if various amino acids appear at the site where 6 amino acids are inserted? investigated. NNS base so that 6 amino acids are inserted between 99-100 (Kabat numbering) in CDR3 of the CE115HA340 sequence (SEQ ID NO: 59), which has higher CD3ε binding activity than CE115HA000, since a decrease in binding activity was expected. Primers were designed using The antibody molecule was expressed as a One arm antibody. Specifically, the above-mentioned H chain including the above-mentioned modification, Kn010G3 (SEQ ID NO: 56), and a sequence in which GLS3000 (SEQ ID NO: 53) and Kappa sequence (SEQ ID NO: 55) are linked as the L chain are employed. Expression purification was performed according to Reference Example 1. The obtained modified antibody was evaluated for binding by the method described in (6-3). The results are shown in Table 11. It has been clarified that the binding property to CD3 (CD3ε) is maintained even when various amino acids appear in the extended amino acid site. Furthermore, Table 12 shows the results of evaluating whether or not non-specific binding is enhanced by the method shown in Reference Example 2. As a result, if a large number of amino acids having a positive charge in the side chain are contained in the extended loop of CDR3, the binding to ECM is enhanced, so there are amino acids having three or more positive charges in the side chain in the loop. It was hoped that it would not appear.
実施例6に記載の検討から、CD3と第2の抗原に結合する抗体取得のための抗体ライブラリ(Dual Fab Library)は以下の様にデザインされた。
ステップ1:CD3(CD3ε)結合能が保持されているアミノ酸(CD3結合量がCE115HA000の80%以上であること)を選択
ステップ2:ECM結合が改変前よりもMRAと比較して10倍以内であるアミノ酸を選択
ステップ3:H鎖CDR3の99-100(Kabat numbering)の間に6アミノ酸を挿入する
なお、ステップ1のみでもFabの抗原結合部位が多様化されるため、第2の抗原に結合する抗原結合分子を同定するライブラリになりうる。また、ステップ1と3のみでもFabの抗原結合部位が多様化されるため、第2の抗原に結合する抗原結合分子を同定するライブラリになりうる。ステップ2を経ないライブラリ設計であっても、取得された分子に対してECM結合を測定し、評価することができる。 (6-7) Design and Construction of Dual Fab Library From the study described in Example 6, an antibody library (Dual Fab Library) for obtaining an antibody that binds to CD3 and the second antigen was designed as follows. .
Step 1: Select amino acids that retain CD3 (CD3ε) binding ability (CD3 binding amount is 80% or more of CE115HA000) Step 2: ECM binding within 10 times compared to MRA than before modification Select a certain amino acid Step 3:
(7-1)ヒトIL6Rに結合するFabドメインの取得
実施例6で設計および構築されたDual Fab libraryからヒトIL6Rに対して結合するFabドメイン(抗体断片)を同定した。抗原として、ビオチン標識されたヒトIL6Rを用いて、ヒトIL6Rに対して結合能をもつ抗体断片の濃縮を行った。
構築されたファージディスプレイ用ファージミドを保持した大腸菌からファージ産生が行われた。ファージ産生が行われた大腸菌の培養液に2.5 M NaCl/10%PEGを添加することによって沈殿させたファージの集団をTBSにて希釈することによってファージライブラリ液が得られた。次に、ファージライブラリ液に終濃度4%BSAとなるようにBSAが添加された。パンニング方法として、一般的な方法である磁気ビーズに固定化した抗原を用いたパンニング方法が参照された(J. Immunol. Methods. (2008) 332 (1-2), 2-9、J. Immunol. Methods. (2001) 247 (1-2), 191-203、Biotechnol. Prog. (2002) 18 (2) 212-20、Mol. Cell Proteomics (2003) 2 (2), 61-9)。磁気ビーズとして、NeutrAvidin coated beads(Sera-Mag SpeedBeads NeutrAvidin-coated)もしくはStreptavidin coated beads(Dynabeads M-280 Streptavidin)が用いられた。 [Example 7] Acquisition of Fab domain binding to CD3 and second antigen (IL6R) from Dual Fab Library (7-1) Acquisition of Fab domain binding to human IL6R Dual designed and constructed in Example 6 A Fab domain (antibody fragment) that binds to human IL6R was identified from the Fab library. Using biotin-labeled human IL6R as an antigen, antibody fragments capable of binding to human IL6R were concentrated.
Phage production was performed from E. coli holding the constructed phagemid for phage display. A phage library solution was obtained by diluting a population of phage precipitated by adding 2.5 M NaCl / 10% PEG to the culture solution of Escherichia coli where phage production was performed, with TBS. Next, BSA was added to the phage library solution to a final concentration of 4% BSA. As a panning method, a panning method using an antigen immobilized on magnetic beads, which is a general method, was referred to (J. Immunol. Methods. (2008) 332 (1-2), 2-9, J. Immunol Methods. (2001) 247 (1-2), 191-203, Biotechnol. Prog. (2002) 18 (2) 212-20, Mol. Cell Proteomics (2003) 2 (2), 61-9). NeutrAvidin coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or Streptavidin coated beads (Dynabeads M-280 Streptavidin) were used as magnetic beads.
さらに、一つの大腸菌に対して複数のファージが感染することを防ぐため、5回目のパンニングにより回収されたファージを感染させた大腸菌より調製したファージライブラリ液について、再度100,000倍希釈したファージ液を大腸菌に感染させてシングルコロニーを得た。 Specifically, by adding 250 pmol of biotin-labeled antigen to the prepared phage library solution, the phage library solution was brought into contact with the antigen at room temperature for 60 minutes. Magnetic beads blocked with BSA were added, and the antigen-phage complex was allowed to bind to the magnetic beads for 15 minutes at room temperature. The beads were washed three times with TBST (TBS containing 0.1
Furthermore, in order to prevent multiple phages from infecting a single E. coli, a phage library solution prepared from E. coli infected with the phages recovered by the fifth panning was again diluted with a 100,000-fold phage solution. To obtain a single colony.
上記の方法によって得られた大腸菌のシングルコロニーから、常法(Methods Mol. Biol. (2002) 178, 133-145)に習い、ファージ含有培養上清が回収された。終濃度4%BSAとなるようにBSAが加えられたファージを含有する培養上清が以下の手順でELISAに供された。StreptaWell 96マイクロタイタープレート(Roche)がビオチン標識抗原(ビオチン化CD3εペプチドもしくはビオチン化ヒトIL6R)を含む100μLのPBSにて4℃で一晩もしくは室温で1時間コートされた。当該プレートの各ウェルをPBSTにて洗浄することによって抗原が除かれた後、当該ウェルが1時間以上250μLの4%BSA-TBSにてブロッキングされた。4%BSA-TBSが除かれた各ウェルに調製された培養上清が加えられた当該プレートを室温で1時間静置することによって、ファージを提示する抗体を各ウェルに存在する抗原に結合させた。各ウェルをTBSTにて洗浄後、終濃度4%のBSAとしたTBSによって希釈されたHRP結合抗M13抗体(Amersham Pharmacia Biotech)を添加して1時間インキュベートした。TBSTにて洗浄後、TMB single溶液(ZYMED)が添加された各ウェル中の溶液の発色反応が、硫酸の添加により停止された後、450 nmの吸光度によって当該発色が測定された。その結果を図19に示す。#50および#62クローンはCD3εおよびヒトIL6Rに対して結合性を有することが示された。すなわち、Dual Fab Libraryを用いることで、第2の抗原(実施例7ではヒトIL6R)に対して結合性を示すクローンを選択することが出来た。さらに評価数を増やし、結合性を示すクローンを選定して、IgG化(クローンが持つVHおよびVL配列をヒトH鎖またはL鎖定常領域とそれぞれ連結すること)し、CD3εと第2の抗原(ヒトIL6R)への結合性を評価することができる。さらに、CD3εと第2の抗原(ヒトIL6R)が同時に結合するか否かが実施例3や4に記載の方法や競合法によって調べることができる。競合法は、例えばCD3εへの結合が、抗体単独のときよりも第2の抗原が存在する場合に低減することで、同時に結合しないことが示される。 (7-2) Binding of Fab domain displayed by phage and CD3 or IL6R (phage ELISA method)
According to a conventional method (Methods Mol. Biol. (2002) 178, 133-145), a phage-containing culture supernatant was recovered from a single colony of E. coli obtained by the above method. The culture supernatant containing the phages to which BSA was added to a final concentration of 4% BSA was subjected to ELISA according to the following procedure.
(8-1)ヒトIgAに結合するFabドメインの取得
IgAは体内に豊富に存在する抗体のアイソタイプであり、腸や粘膜面での生体防御に関わる分子として知られており、FcαR(Fc alpha Receptor)に結合することが知られている(J. Pathol. 208: 270 -282, 2006)。
実施例6で設計および構築されたDual Fab libraryからヒトIgAに対して結合するFabドメイン(抗体断片)を同定した。抗原として、ビオチン標識されたヒトIgA(参考実施例3に記載されている。)を用いて、ヒトIgAに対して結合能をもつ抗体断片の濃縮を行った。
構築されたファージディスプレイ用ファージミドを保持した大腸菌からファージ産生が行われた。ファージ産生が行われた大腸菌の培養液に2.5 M NaCl/10%PEGを添加することによって沈殿させたファージの集団をTBSにて希釈することによってファージライブラリ液が得られた。次に、ファージライブラリ液に終濃度4%BSAとなるようにBSAが添加された。パンニング方法として、一般的な方法である磁気ビーズに固定化した抗原を用いたパンニング方法が参照された(J. Immunol. Methods. (2008) 332 (1-2), 2-9、J. Immunol. Methods. (2001) 247 (1-2), 191-203、Biotechnol. Prog. (2002) 18 (2) 212-20、Mol. Cell Proteomics (2003) 2 (2), 61-9)。磁気ビーズとして、NeutrAvidin coated beads(Sera-Mag SpeedBeads NeutrAvidin-coated)もしくはStreptavidin coated beads(Dynabeads M-280 Streptavidin)が用いられた。
具体的には、調製されたファージライブラリ液に250 pmolのビオチン標識抗原を加えることによって、当該ファージライブラリ液を室温にて60分間抗原と接触させた。BSAでブロッキングされた磁気ビーズが加えられ、抗原とファージとの複合体を磁気ビーズと室温にて15分間結合させた。ビーズはTBST(0.1%Tween20を含有するTBS, TBSはTaKaRa社製)にて3回洗浄された後、1 mLのTBSにてさらに2回洗浄された。その後、0.5 mLの1 mg/mLのトリプシンが加えられたビーズは室温で15分懸濁された後、即座に磁気スタンドを用いてビーズが分離され、ファージ溶液が回収された。回収されたファージ溶液が、対数増殖期(OD600が0.4-0.5)となった10 mLの大腸菌株ER2738に添加された。37℃で1時間緩やかに上記大腸菌の攪拌培養を行うことによって、ファージを大腸菌に感染させた。感染させた大腸菌は、225 mm x 225 mmのプレートへ播種された。次に、播種された大腸菌の培養液からファージを回収することによって、ファージライブラリ液が調製された。このサイクルをパンニングと呼び、4回繰り返した。なお2回目以降のパンニングでは、ヒトIgAは40pmolとした。 [Example 8] Acquisition of Fab domain binding to CD3 and second antigen (human IgA) from Dual Fab Library (8-1) Acquisition of Fab domain binding to human IgA IgA is an abundant antibody in the body It is known as a molecule involved in biological defense in the intestine and mucosa, and is known to bind to FcαR (Fc alpha Receptor) (J. Pathol. 208: 270 -282, 2006) .
The Fab domain (antibody fragment) that binds to human IgA was identified from the Dual Fab library designed and constructed in Example 6. The antibody fragment capable of binding to human IgA was concentrated using biotin-labeled human IgA (described in Reference Example 3) as an antigen.
Phage production was performed from E. coli holding the constructed phagemid for phage display. A phage library solution was obtained by diluting a population of phage precipitated by adding 2.5 M NaCl / 10% PEG to the culture solution of Escherichia coli where phage production was performed, with TBS. Next, BSA was added to the phage library solution to a final concentration of 4% BSA. As a panning method, a panning method using an antigen immobilized on magnetic beads, which is a general method, was referred to (J. Immunol. Methods. (2008) 332 (1-2), 2-9, J. Immunol Methods. (2001) 247 (1-2), 191-203, Biotechnol. Prog. (2002) 18 (2) 212-20, Mol. Cell Proteomics (2003) 2 (2), 61-9). NeutrAvidin coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or Streptavidin coated beads (Dynabeads M-280 Streptavidin) were used as magnetic beads.
Specifically, by adding 250 pmol of biotin-labeled antigen to the prepared phage library solution, the phage library solution was brought into contact with the antigen at room temperature for 60 minutes. Magnetic beads blocked with BSA were added, and the antigen-phage complex was allowed to bind to the magnetic beads for 15 minutes at room temperature. The beads were washed three times with TBST (TBS containing 0.1
上記の方法によって得られた大腸菌のシングルコロニーから、常法(Methods Mol. Biol. (2002) 178, 133-145)に習い、ファージ含有培養上清が回収された。終濃度4%BSAとなるようにBSAが加えられたファージを含有する培養上清が以下の手順でELISAに供された。StreptaWell 96マイクロタイタープレート(Roche)がビオチン標識抗原(ビオチン標識CD3εペプチドもしくはビオチン標識ヒトIgA,参考実施例3)を含む100μLのPBSにて4℃で一晩もしくは室温で1時間コートされた。当該プレートの各ウェルをPBSTにて洗浄することによって抗原が除かれた後、当該ウェルが1時間以上250μLの0.1xTBS/150mM NaCl/0.02% Skim Milkにてブロッキングされた。0.1xTBS/150mM NaCl/0.02% Skim Milkが除かれた各ウェルに調製された培養上清が加えられた当該プレートを室温で1時間静置することによって、ファージが提示する抗体を各ウェルに存在する抗原に結合させた。各ウェルを0.1xTBS/150mM NaCl/0.01% Tween20にて洗浄後、0.1xTBS/150mM NaCl/0.01% Tween20によって希釈されたHRP結合抗M13抗体(Amersham Pharmacia Biotech)を添加して1時間インキュベートした。TBSTにて洗浄後、TMB single溶液(ZYMED)が添加された各ウェル中の溶液の発色反応が、硫酸の添加により停止された後、450 nmの吸光度によって当該発色が測定された。その結果を図20に示す。図20に示すように、CD3およびヒトIgAに結合するクローンが存在することが示され、Dual Fab Libraryを用いることで、第2の抗原(実施例8ではヒトIgA)に対して結合性を示すクローンを選択することが出来た。 (8-2) Binding of Fab-displayed Fab domain to CD3 or human IgA From a single colony of Escherichia coli obtained by the above-mentioned method, it was learned from conventional methods (Methods Mol. Biol. (2002) 178, 133-145). The phage-containing culture supernatant was collected. The culture supernatant containing the phages to which BSA was added to a final concentration of 4% BSA was subjected to ELISA according to the following procedure.
(8-2)でCD3とヒトIgAに結合することが示されたクローンに対して、その配列を有する大腸菌からDual Fab LibraryのH鎖に特異的に結合するプライマーを用いて、PCR法によってVH断片を増幅した。増幅したVH断片は参考実施例1の方法でpE22Hhが組み込まれた動物細胞発現用のプラスミドに組み込まれ、実施例6(6-2)と同様にOne arm抗体として発現・精製された。クローン名とH鎖配列の配列番号は表15に示されている。すなわち、表15に示されたH鎖とKn010G3(配列番号:56)とL鎖としてGLS3000(配列番号:53)とKappa配列(配列番号:55)を連結した配列を採用し、参考実施例1に従って発現精製が行われた。 (8-3) Binding of IgG having the obtained Fab domain to CD3 or human IgA (8-2) From a clone that was shown to bind to CD3 and human IgA, E. coli having the sequence was dually tested. A VH fragment was amplified by PCR using a primer that specifically binds to the Fab Library H chain. The amplified VH fragment was incorporated into an animal cell expression plasmid into which pE22Hh had been incorporated by the method of Reference Example 1, and expressed and purified as a One arm antibody in the same manner as in Example 6 (6-2). The clone name and the SEQ ID NO of the H chain sequence are shown in Table 15. That is, the sequence shown in Table 15 in which GLS3000 (SEQ ID NO: 53) and Kappa sequence (SEQ ID NO: 55) are linked as the H chain, Kn010G3 (SEQ ID NO: 56), and L chain is employed. Expression purification was performed according to
この結果から、Dual Fab libraryから、第2の抗原と結合する抗体が取得可能であり、通常ファージライブラリを用いたパンニングではFabドメインのみであるが、Fc領域を含むIgGとなっても結合が認められるクローンの濃縮が可能であることが示された。したがって、Dual Fab Libraryは、CD3との結合能を保持しながら第2の抗原との結合能を有するFabドメインを取得することができるLibraryだといえた。 The result is shown in FIG. When clones that were found to be bound by phage ELISA were converted to IgG, including those containing some amino acid mutations, the sequences that were found to be bound by phage ELISA should bind to CD3ε and human IgA even if they were IgG. It has been shown.
From this result, it is possible to obtain an antibody that binds to the second antigen from the Dual Fab library. Normally, panning using a phage library has only a Fab domain, but binding to an IgG containing an Fc region is recognized. It was shown that enrichment of the resulting clones is possible. Therefore, it can be said that the Dual Fab Library is a library capable of obtaining a Fab domain having the ability to bind to the second antigen while retaining the ability to bind to CD3.
(8-3)において、Dual Fab Libraryから得られたクローンがIgGとなっても結合を有することが示された。次に、得られたIgGがCD3(CD3ε)及びヒトIgAと同時に結合するかを競合法(電気化学発光法(ECL法))で判定した。CD3(CD3ε)及びヒトIgAと同時に結合する場合は、IgAと結合している抗体にCD3(CD3ε)を加えてもECLシグナルは変化しないが、同時結合できない場合には、CD3(CD3ε)を加えると一部の抗体がCD3(CD3ε)と結合してECLシグナルが低下するはずである。 (8-4) Evaluation of simultaneous binding of the obtained Fab domain IgG with CD3 (CD3ε) and human IgA In (8-3), the clone obtained from Dual Fab Library has binding even if it becomes IgG It was shown that. Next, it was determined by competitive method (electrochemiluminescence method (ECL method)) whether or not the obtained IgG binds simultaneously with CD3 (CD3ε) and human IgA. When binding simultaneously with CD3 (CD3ε) and human IgA, adding CD3 (CD3ε) to the antibody binding to IgA does not change the ECL signal, but if simultaneous binding is not possible, add CD3 (CD3ε) And some antibodies should bind to CD3 (CD3ε) and decrease ECL signal.
実施例8において、CD3εおよびヒトIgAに結合し、CD3εとヒトIgAが同時に結合しないDual Fab分子が得られることが示された。さらに第3の抗原と結合する抗原結合ドメインを付加することも当業者公知の方法で実施可能である。
近年、癌抗原の一つであるEGFRに結合するように改変されたIgA分子がEGFRを発現している癌細胞に細胞死を誘導することが示された(J Immunol 2007; 179:2936-2943)。このメカニズムとしてIgAのレセプターであるFcαRがPolymorphonuclear cellに発現しており、癌細胞にオートファジーを誘導することが報告された(J Immunol 2011; 187:726-732)。本実施例でCD3とIgAに結合するDual Fab分子が構築できることが明らかになったが、IgAを介してFcαRと結合する分子を当業者公知の方法(例えばELISAやECL法)で探索すれば、FcαRを介した抗腫瘍効果が期待できる。すなわち、本Dual Fabは、任意の第3の抗原が発現している細胞に、CD3εとの結合でT細胞による細胞傷害活性と、IgAとの結合を介してFcαRが発現している細胞による細胞傷害活性の両方を誘導することが可能であり、強い細胞傷害活性が期待できる。 (8-5) CD3 / Human IgA Dual Fab Molecule In Example 8, it was shown that a Dual Fab molecule that binds to CD3ε and human IgA and does not bind CD3ε and human IgA simultaneously is obtained. Furthermore, it is possible to add an antigen-binding domain that binds to the third antigen by a method known to those skilled in the art.
Recently, IgA molecules modified to bind to EGFR, one of the cancer antigens, were shown to induce cell death in EGFR-expressing cancer cells (J Immunol 2007; 179: 2936-2943 ). It has been reported that FcαR, an IgA receptor, is expressed in polymorphonuclear cells and induces autophagy in cancer cells (J Immunol 2011; 187: 726-732). In this example, it was clarified that a dual Fab molecule that binds to CD3 and IgA can be constructed. However, if a molecule that binds to FcαR via IgA is searched by a method known to those skilled in the art (for example, ELISA or ECL method), Antitumor effects can be expected through FcαR. In other words, this dual Fab can be applied to cells expressing an arbitrary third antigen, to cells produced by cells expressing FcαR via binding to CD3ε and T cells by binding to CD3ε and IgA. Both cytotoxic activities can be induced, and a strong cytotoxic activity can be expected.
(9-1)ヒトCD154に結合するFabドメインの取得
実施例6で設計および構築されたDual Fab libraryからヒトCD154に対して結合するFabドメイン(抗体断片)を同定した。抗原として、ビオチン標識されたヒトCD154を用いて、ヒトCD154に対して結合能をもつ抗体断片の濃縮を行った。
構築されたファージディスプレイ用ファージミドを保持した大腸菌からファージ産生が行われた。ファージ産生が行われた大腸菌の培養液に2.5 M NaCl/10%PEGを添加することによって沈殿させたファージの集団をTBSにて希釈することによってファージライブラリ液が得られた。次に、ファージライブラリ液に終濃度4%BSAとなるようにBSAが添加された。パンニング方法として、一般的な方法である磁気ビーズに固定化した抗原を用いたパンニング方法が参照された(J. Immunol. Methods. (2008) 332 (1-2), 2-9、J. Immunol. Methods. (2001) 247 (1-2), 191-203、Biotechnol. Prog. (2002) 18 (2) 212-20、Mol. Cell Proteomics (2003) 2 (2), 61-9)。磁気ビーズとして、NeutrAvidin coated beads(Sera-Mag SpeedBeads NeutrAvidin-coated)もしくはStreptavidin coated beads(Dynabeads M-280 Streptavidin)が用いられた。
具体的には、調製されたファージライブラリ液に250 pmolのビオチン標識抗原を加えることによって、当該ファージライブラリ液を室温にて60分間抗原と接触させた。BSAでブロッキングされた磁気ビーズが加えられ、抗原とファージとの複合体を磁気ビーズと室温にて15分間結合させた。ビーズはTBST(0.1%Tween20を含有するTBS, TBSはTaKaRa社製)にて3回洗浄された後、1 mLのTBSにてさらに2回洗浄された。その後、0.5 mLの1 mg/mLのトリプシンが加えられたビーズは室温で15分懸濁された後、即座に磁気スタンドを用いてビーズが分離され、ファージ溶液が回収された。回収されたファージ溶液が、対数増殖期(OD600が0.4-0.5)となった10 mLの大腸菌株ER2738に添加された。37℃で1時間緩やかに上記大腸菌の攪拌培養を行うことによって、ファージを大腸菌に感染させた。感染させた大腸菌は、225 mm x 225 mmのプレートへ播種された。次に、播種された大腸菌の培養液からファージを回収することによって、ファージライブラリ液が調製された。このサイクルをパンニングと呼び、5回繰り返した。なお2回目以降のパンニングでは、ヒトCD154は40pmolとした。 [Example 9] Acquisition of Fab domain binding to CD3 and second antigen (human CD154) from Dual Fab Library (9-1) Acquisition of Fab domain binding to human CD154 Designed and constructed in Example 6 A Fab domain (antibody fragment) that binds to human CD154 was identified from the Dual Fab library. Using biotin-labeled human CD154 as an antigen, antibody fragments capable of binding to human CD154 were concentrated.
Phage production was performed from E. coli holding the constructed phagemid for phage display. A phage library solution was obtained by diluting a population of phage precipitated by adding 2.5 M NaCl / 10% PEG to the culture solution of Escherichia coli where phage production was performed, with TBS. Next, BSA was added to the phage library solution to a final concentration of 4% BSA. As a panning method, a panning method using an antigen immobilized on magnetic beads, which is a general method, was referred to (J. Immunol. Methods. (2008) 332 (1-2), 2-9, J. Immunol Methods. (2001) 247 (1-2), 191-203, Biotechnol. Prog. (2002) 18 (2) 212-20, Mol. Cell Proteomics (2003) 2 (2), 61-9). NeutrAvidin coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or Streptavidin coated beads (Dynabeads M-280 Streptavidin) were used as magnetic beads.
Specifically, by adding 250 pmol of biotin-labeled antigen to the prepared phage library solution, the phage library solution was brought into contact with the antigen at room temperature for 60 minutes. Magnetic beads blocked with BSA were added, and the antigen-phage complex was allowed to bind to the magnetic beads for 15 minutes at room temperature. The beads were washed three times with TBST (TBS containing 0.1
上記の方法によって得られた大腸菌のシングルコロニーから、常法(Methods Mol. Biol. (2002) 178, 133-145)に習い、ファージ含有培養上清が回収された。終濃度4%BSAとなるようにBSAが加えられたファージを含有する培養上清が以下の手順でELISAに供された。StreptaWell 96マイクロタイタープレート(Roche)がビオチン標識抗原(ビオチン標識CD3εペプチド、ビオチン標識CD154)を含む100μLのPBSにて4℃で一晩もしくは室温で1時間コートされた。当該プレートの各ウェルをPBSTにて洗浄することによって抗原が除かれた後、当該ウェルが1時間以上250μLの0.1xTBS/150mM NaCl/0.02% Skim Milkにてブロッキングされた。0.1xTBS/150mM NaCl /0.02% Skim Milkが除かれた各ウェルに調製された培養上清が加えられた当該プレートを室温で1時間静置することによって、ファージを提示する抗体を各ウェルに存在する抗原に結合させた。各ウェルを0.1xTBS/150mM NaCl/0.01% Tween20にて洗浄後、0.1xTBS/150mM NaCl/0.01% Tween20によって希釈されたHRP結合抗M13抗体(Amersham Pharmacia Biotech)を添加して1時間インキュベートした。TBSTにて洗浄後、TMB single溶液(ZYMED)が添加された各ウェル中の溶液の発色反応が、硫酸の添加により停止された後、450 nmの吸光度によって当該発色が測定された。その結果を図23に示す。図23に示すように、CD3およびCD154に結合するクローンが存在することが示され、Dual Fab Libraryを用いることで、第2の抗原(実施例9ではヒトCD154)に対して結合性を示すクローンを選択することが出来た。また、実施例7,8,9で示したように異なる3つの抗原に対して、結合Fabドメインが取得できることから、Dual Fab Libraryが第2の抗原と結合する分子を取得するためのライブラリとして機能することが示された。 (9-2) Binding of Fab-displayed Fab domain to CD3 or human CD154 From a single colony of Escherichia coli obtained by the above method, learn from conventional methods (Methods Mol. Biol. (2002) 178, 133-145) The phage-containing culture supernatant was collected. The culture supernatant containing the phages to which BSA was added to a final concentration of 4% BSA was subjected to ELISA according to the following procedure.
(9-2)でCD3とヒトCD154に結合することが示されたクローンに対して、その配列を有する大腸菌からDual Fab LibraryのH鎖に特異的に結合するプライマーを用いて、PCRによってVH断片を増幅した。増幅したVH断片は参考実施例1の方法でpE22Hhが組み込まれた動物細胞発現用のプラスミドに組み込まれ、実施例6(6-2)と同様にOne arm抗体として発現・精製された。取得された配列名とH鎖配列の配列番号は表16に記載されている。具体的には、表16に記載されているH鎖とKn010G3(配列番号:56)とL鎖としてGLS3000(配列番号:53)とKappa配列(配列番号:55)を連結した配列を採用し、参考実施例1に従って発現精製が行われた。 (9-3) Binding of IgG having Fab domain obtained to CD3 or human CD154 (9-2) From a clone shown to bind to CD3 and human CD154 in (9-2), from E. coli having the sequence to Dual A VH fragment was amplified by PCR using a primer that specifically binds to the Fab Library H chain. The amplified VH fragment was incorporated into an animal cell expression plasmid into which pE22Hh had been incorporated by the method of Reference Example 1, and expressed and purified as a One arm antibody in the same manner as in Example 6 (6-2). The obtained sequence names and the sequence numbers of the H chain sequences are shown in Table 16. Specifically, a sequence in which GLS3000 (SEQ ID NO: 53) and Kappa sequence (SEQ ID NO: 55) are linked as H chain, Kn010G3 (SEQ ID NO: 56) and L chain described in Table 16 is adopted, Expression purification was performed according to Reference Example 1.
この結果から、Dual Fab libraryから、第2の抗原と結合する抗体が取得可能であり、通常ファージライブラリを用いたパンニングではFabドメインのみであるが、Fc領域を含むIgGとなっても結合が認められるクローンの濃縮がヒトIgAだけでなくヒトCD154でも可能であることが示された。したがって、Dual Fab Libraryは、CD3との結合能を保持しながら第2の抗原との結合能を有するFabドメインを取得することができるLibraryだといえる。 The result is shown in FIG. When clones that were found to be bound by phage ELISA were converted to IgG, including those containing some amino acid mutations, the sequences that were found to be bound by phage ELISA would bind to CD3ε and human CD154 even if they became IgG. It has been shown.
From this result, it is possible to obtain an antibody that binds to the second antigen from the Dual Fab library. Normally, panning using a phage library has only a Fab domain, but binding to an IgG containing an Fc region is recognized. It was shown that enrichment of the resulting clones is possible not only with human IgA but also with human CD154. Therefore, it can be said that the Dual Fab Library is a library capable of obtaining a Fab domain having the ability to bind to the second antigen while retaining the ability to bind to CD3.
(9-3)において、Dual Fab Libraryから得られたクローンがIgGとなっても結合を有することが示された。次に、得られたIgGがCD3及びヒトCD154と同時に結合するかを競合法(電気化学発光法(ECL法))で判定した。CD3及びヒトCD154と同時に結合する場合は、CD154と結合している抗体にCD3を加えてもECLシグナルは変化しないが、同時結合できない場合には、CD3を加えると一部の抗体がCD3と結合してECLシグナルが低下するはずである。 (9-4) Evaluation of simultaneous binding of the obtained Fab domain IgG with CD3ε and human CD154 (9-3) shows that the clone obtained from the Dual Fab Library has binding even if it becomes IgG It was done. Next, it was determined by a competition method (electrochemiluminescence method (ECL method)) whether the obtained IgG binds simultaneously with CD3 and human CD154. When binding to CD3 and human CD154 simultaneously, adding CD3 to the antibody that binds to CD154 does not change the ECL signal, but if simultaneous binding is not possible, adding CD3 causes some antibodies to bind to CD3. Then the ECL signal should drop.
実施例9において、CD3εおよびヒトCD154に結合し、CD3εとヒトCD154が同時に結合しないDual Fab分子が得られることが示された。さらに第3の抗原と結合する抗原結合ドメインを付加することも当業者公知の方法で実施可能である。
近年、CD154のレセプターであるCD40のアゴニスト抗体が癌抗原反応性のT細胞を移入する方法において抗腫瘍活性を増強することが示された(J Immunother. 2012 Apr;35(3):276-82.)。本実施例でCD3とCD154に結合するDual Fab分子が構築できることが明らかになったが、CD154を介してCD40に対してアゴニスト活性を示す抗体を選定すれば、CD40を介した抗腫瘍効果が期待できる。すなわち、本Dual Fabは、任意の第3の抗原が発現している細胞に、CD3εとの結合でT細胞による細胞傷害活性と、CD154との結合を介してCD40のアゴニストシグナルによる抗腫瘍効果の増強が期待できる。 (9-5) CD3 / Human CD154 Dual Fab Molecule In Example 9, it was shown that a Dual Fab molecule that binds to CD3ε and human CD154 and does not bind CD3ε and human CD154 at the same time is obtained. Furthermore, it is possible to add an antigen-binding domain that binds to the third antigen by a method known to those skilled in the art.
Recently, it has been shown that an agonist antibody of CD40, which is a receptor for CD154, enhances antitumor activity in a method of transferring cancer antigen-reactive T cells (J Immunother. 2012 Apr; 35 (3): 276-82 .). In this example, it was clarified that a dual Fab molecule that binds to CD3 and CD154 can be constructed. However, if an antibody exhibiting agonist activity against CD40 is selected via CD154, antitumor effect via CD40 is expected. it can. That is, this dual Fab is capable of inhibiting the cytotoxic activity of T cells by binding to CD3ε and the antitumor effect of CD40 agonistic signals through binding to CD154 on cells expressing an arbitrary third antigen. An increase can be expected.
〔参考実施例1〕抗体の発現ベクターの作製および抗体の発現と精製
アミノ酸置換の導入はQuikChange Site-Directed Mutagenesis Kit(Stratagene)、PCRまたはIn fusion Advantage PCR cloning kit (TAKARA)等を用いて当業者公知の方法で行い、発現ベクターを構築した。得られた発現ベクターの塩基配列は当業者公知の方法で決定した。作製したプラスミドをヒト胎児腎癌細胞由来HEK293H株(Invitrogen)、またはFreeStyle293細胞(Invitrogen社)に、一過性に導入し、抗体の発現を行った。得られた培養上清から、rProtein A SepharoseTM Fast Flow(GEヘルスケア)を用いて当業者公知の方法で、抗体を精製した。精製抗体濃度は、分光光度計を用いて280 nmでの吸光度を測定し、得られた値からPACE法により算出された吸光係数を用いて抗体濃度を算出した(Protein Science 1995 ; 4 : 2411-2423)。 [Reference Example]
[Reference Example 1] Preparation of antibody expression vector and expression and purification of antibody Introduction of amino acid substitution is performed by those skilled in the art using QuikChange Site-Directed Mutagenesis Kit (Stratagene), PCR or In fusion Advantage PCR cloning kit (TAKARA), etc. An expression vector was constructed by a known method. The base sequence of the obtained expression vector was determined by a method known to those skilled in the art. The prepared plasmid was transiently introduced into a human fetal kidney cancer cell-derived HEK293H strain (Invitrogen) or FreeStyle293 cells (Invitrogen) to express the antibody. The antibody was purified from the obtained culture supernatant by a method known to those skilled in the art using rProtein A Sepharose ™ Fast Flow (GE Healthcare). The purified antibody concentration was determined by measuring the absorbance at 280 nm using a spectrophotometer, and calculating the antibody concentration using the extinction coefficient calculated by the PACE method from the obtained value (Protein Science 1995; 4: 2411- 2423).
抗体のECM(Extracellular matrix)への結合評価はWO2012093704A1を参考に以下の手順で実施された。ECM Phenol red free(BD Matrigel #356237)をTBSで2mg/mLに希釈し、氷上で冷やしたECL測定用プレート(L15XB-3、MSD high bind)の各ウエルの真ん中に5μL滴下した。その後、プレートシールで蓋をして4℃で一晩静置した。ECMを固相化したプレートを室温に戻し、ECL Blocking Buffer(PBSに0.5%BSAおよび0.05%Tween 20を加えたもの)150μLを各ウエルに加えて、室温で静置2時間以上もしくは4℃で一晩静置した。次に、PBS-T(PBSに0.05%Tween 20を加えたもの)を用いて抗体サンプルを9μg/mLに希釈した。2次抗体をECLDB(PBSに0.1%BSAおよび0.01%Tween 20を加えたもの)で2μg/mLに希釈し、ECLDBが10μLずつ各ウエルに分注されている丸底プレートへ、抗体溶液20μL、2次抗体溶液30μLを加えて、遮光下室温で1時間攪拌した。ECL Bloking Bufferが入ったECMプレートから、ECL Blocking Bufferを転倒除去し、このプレートへ、前述の抗体・二次抗体の混合溶液を50μLずつ加えた。その後遮光下室温で1時間静置した。サンプルを転倒除去後、READ buffer(MSD)を各ウェルに150μLずつ加え、Sector Imager 2400(MSD)でsulfo-tagの発光シグナルを検出した。 [Reference Example 2] Evaluation of antibody binding to ECM (Extracellular matrix) The evaluation of antibody binding to ECM (Extracellular matrix) was performed by the following procedure with reference to WO2012093704A1. ECM Phenol red free (BD Matrigel # 356237) was diluted with TBS to 2 mg / mL, and 5 μL was dropped in the middle of each well of an ECL measurement plate (L15XB-3, MSD high bind) cooled on ice. Then, it was covered with a plate seal and allowed to stand at 4 ° C. overnight. Return the ECM-immobilized plate to room temperature, add 150 μL of ECL Blocking Buffer (PBS with 0.5% BSA and 0.05% Tween 20) to each well, and allow to stand at room temperature for at least 2 hours or at 4 ° C. Let stand overnight. Next, the antibody sample was diluted to 9 μg / mL using PBS-T (0.05
ヒトIgAとして天然に存在するヒトIgA配列のうちFc部分を用いた(ヒトIgA-Fc)。ヒトIgA-FcのC末端にbiotinを付加するためビオチンリガーゼによってビオチンが付加される特異的な配列(AviTag配列、配列番号:79)をコードする遺伝子断片をリンカーを介して連結させた。ヒトIgA-FcとAviTag配列が連結されたタンパク質(配列番号:80)をコードする遺伝子断片を動物細胞発現用ベクターに組み込み、構築されたプラスミドベクターを293Fectin (Invitrogen)を用いてFreeStyle293細胞 (Invitrogen)に導入した。このときEBNA1(配列番号:81)を発現する遺伝子およびビオチンリガーゼ(BirA、配列番号:82)を発現する遺伝子を同時に導入し、さらにヒトIgA-Fcをビオチン標識する目的でビオチンを添加した。前述の手順に従って遺伝子が導入された細胞を37℃、8% CO2で6日間培養し、目的のタンパク質を培養上清中に分泌させた。
目的のヒトIgA-Fcを含む細胞培養液を0.22μmボトルトップフィルターでろ過し、培養上清を得た。20 mM Tris-HCl, pH7.4で平衡化されたHiTrap Q HP (GEヘルスケア)に、同溶液で希釈された培養上清をアプライし、NaClの濃度勾配により目的のヒトIgA-Fcを溶出させた。次に、50 mM Tris-HCl, pH8.0で平衡化されたSoftLink Avidin カラム(Promega)に、同溶液で希釈された前記HiTrap Q HP溶出液をアプライし、5 mM ビオチン, 150 mM NaCl, 50 mM Tris-HCl, pH8.0で目的のヒトIgA-Fcを溶出させた。その後、Superdex200(GEヘルスケア)を用いたゲルろ過クロマトグラフィーによって、目的外の不純物である会合体を除去し、バッファーが20 mM Histidine-HCl, 150 mM NaCl, pH6.0に置換された精製ヒトIgA-Fcを得た。 [Reference Example 3] Preparation of human IgA The Fc portion of the human IgA sequence naturally present as human IgA was used (human IgA-Fc). In order to add biotin to the C-terminus of human IgA-Fc, a gene fragment encoding a specific sequence (AviTag sequence, SEQ ID NO: 79) to which biotin is added by biotin ligase was ligated via a linker. A gene fragment encoding a protein (SEQ ID NO: 80) in which human IgA-Fc and AviTag sequences are linked is incorporated into an animal cell expression vector, and the constructed plasmid vector is used in FreeStyle293 cells (Invitrogen) using 293Fectin (Invitrogen). Introduced. At this time, a gene expressing EBNA1 (SEQ ID NO: 81) and a gene expressing biotin ligase (BirA, SEQ ID NO: 82) were simultaneously introduced, and biotin was added for the purpose of biotin labeling human IgA-Fc. Cells into which the gene had been introduced were cultured for 6 days at 37 ° C. and 8% CO 2 according to the procedure described above, and the target protein was secreted into the culture supernatant.
The cell culture solution containing the target human IgA-Fc was filtered through a 0.22 μm bottle top filter to obtain a culture supernatant. Apply the culture supernatant diluted with the same solution to HiTrap Q HP (GE Healthcare) equilibrated with 20 mM Tris-HCl, pH 7.4, and elute the target human IgA-Fc with NaCl concentration gradient I let you. Next, the HiTrap Q HP eluate diluted with the same solution was applied to a SoftLink Avidin column (Promega) equilibrated with 50 mM Tris-HCl, pH 8.0, and 5 mM biotin, 150 mM NaCl, 50 The target human IgA-Fc was eluted with mM Tris-HCl, pH 8.0. Subsequently, purified humans were removed by gel filtration chromatography using Superdex200 (GE Healthcare) to remove aggregates, which were impurities of no interest, and the buffer was replaced with 20 mM Histidine-HCl, 150 mM NaCl, pH 6.0. IgA-Fc was obtained.
Claims (30)
- 第1の抗原および該第1の抗原とは異なる第2の抗原に結合することができるが、第1の抗原と第2の抗原に同時には結合しない、抗体の可変領域、並びに
該第1の抗原および第2の抗原とは異なる第3の抗原に結合する可変領域
を含む、抗原結合分子。 A variable region of an antibody capable of binding to a first antigen and a second antigen different from the first antigen, but not simultaneously binding to the first antigen and the second antigen, and the first antigen An antigen-binding molecule comprising a variable region that binds to a third antigen different from the antigen and the second antigen. - 第1の抗原および該第1の抗原とは異なる第2の抗原に結合することができるが、第1の抗原と第2の抗原に同時には結合しないように、重鎖可変領域のアミノ酸が改変された抗体の可変領域を含む、抗原結合分子。 The amino acid of the heavy chain variable region is modified so that it can bind to the first antigen and a second antigen different from the first antigen, but does not bind to the first antigen and the second antigen simultaneously. An antigen-binding molecule comprising a variable region of a prepared antibody.
- 第1の抗原と第2の抗原に同時には結合しない可変領域が、それぞれ異なる細胞上で発現している第1の抗原と第2の抗原に同時には結合しない可変領域である、請求項1又は2に記載の抗原結合分子。 The variable region that does not bind to the first antigen and the second antigen at the same time is a variable region that does not bind to the first antigen and the second antigen that are expressed on different cells, respectively. 3. The antigen-binding molecule according to 2.
- さらに、抗体のFc領域を含む、請求項1から3のいずれかに記載の抗原結合分子。 The antigen-binding molecule according to any one of claims 1 to 3, further comprising an Fc region of the antibody.
- Fc領域のFcγRに対する結合活性が、天然型ヒトIgG1抗体のFc領域と比較して低下しているFc領域である、請求項4に記載の抗原結合分子。 The antigen-binding molecule according to claim 4, wherein the Fc region has a reduced Fc region binding activity to FcγR compared to the Fc region of a natural human IgG1 antibody.
- 多重特異性抗体である、請求項1から5のいずれかに記載の抗原結合分子。 The antigen-binding molecule according to any one of claims 1 to 5, which is a multispecific antibody.
- 第1の抗原と第2の抗原に結合することができる抗体の可変領域が、少なくとも1つのアミノ酸の改変が導入されている可変領域である、請求項1から6のいずれかに記載の抗原結合分子。 The antigen binding according to any one of claims 1 to 6, wherein the variable region of an antibody capable of binding to the first antigen and the second antigen is a variable region into which at least one amino acid modification has been introduced. molecule.
- 前記改変が、少なくとも1つのアミノ酸の置換又は挿入である、請求項7に記載の抗原結合分子。 The antigen-binding molecule according to claim 7, wherein the modification is substitution or insertion of at least one amino acid.
- 前記改変が、第1の抗原に結合する可変領域のアミノ酸配列の一部の、第2の抗原に結合するアミノ酸配列への置換、又は、第1の抗原に結合する可変領域のアミノ酸配列への、第2の抗原に結合するアミノ酸配列の挿入である、請求項7又は8に記載の抗原結合分子。 The modification is the replacement of a part of the amino acid sequence of the variable region that binds to the first antigen with the amino acid sequence that binds to the second antigen, or the amino acid sequence of the variable region that binds to the first antigen. The antigen-binding molecule according to claim 7 or 8, which is an insertion of an amino acid sequence that binds to a second antigen.
- 挿入されるアミノ酸の数が1~25個である、請求項8又は9に記載の抗原結合分子。 The antigen-binding molecule according to claim 8 or 9, wherein the number of inserted amino acids is 1 to 25.
- 改変されるアミノ酸が、抗体の可変領域のCDR1、CDR2、CDR3又はFR3領域のアミノ酸である、請求項7から10のいずれかに記載の抗原結合分子。 The antigen-binding molecule according to any one of claims 7 to 10, wherein the amino acid to be modified is an amino acid in the CDR1, CDR2, CDR3, or FR3 region of the variable region of the antibody.
- 改変されるアミノ酸が、ループ領域のアミノ酸である、請求項7から11のいずかに記載の抗原結合分子。 The antigen-binding molecule according to any one of claims 7 to 11, wherein the amino acid to be modified is a loop region amino acid.
- 改変されるアミノ酸が、抗体のH鎖可変領域のKabatナンバリング31~35、50~65、71~74及び95~102、L鎖可変領域のKabatナンバリング24~34、50~56及び89~97から選ばれる少なくとも1つのアミノ酸である、請求項7から11のいずれかに記載の抗原結合分子。 The amino acids to be modified are from Kabat numbering 31-35, 50-65, 71-74 and 95-102 of the heavy chain variable region of the antibody, and Kabat numbering 24-34, 50-56 and 89-97 of the light chain variable region. The antigen-binding molecule according to any one of claims 7 to 11, which is at least one amino acid selected.
- 第1の抗原又は第2の抗原のいずれか1つが、T細胞表面に特異的に発現している分子であり、他方の抗原が、T細胞又は他の免疫細胞表面に発現している分子である、請求項1から13のいずれかに記載の抗原結合分子。 Either one of the first antigen and the second antigen is a molecule that is specifically expressed on the surface of T cells, and the other antigen is a molecule that is expressed on the surface of T cells or other immune cells. The antigen-binding molecule according to any one of claims 1 to 13.
- 第1の抗原又は第2の抗原のいずれか1つがCD3であり、他方の抗原がFcγR、TLR、レクチン、IgA、免疫チェックポイント分子、TNFスーパーファミリー分子、TNFRスーパーファミリー分子又はNKレセプター分子である、請求項14に記載の抗原結合分子。 Either one of the first antigen or the second antigen is CD3, and the other antigen is FcγR, TLR, lectin, IgA, immune checkpoint molecule, TNF superfamily molecule, TNFR superfamily molecule or NK receptor molecule The antigen-binding molecule according to claim 14.
- 第3の抗原が、がん組織特異的に発現している分子である、請求項14又は15に記載の抗原結合分子。 The antigen-binding molecule according to claim 14 or 15, wherein the third antigen is a molecule expressed specifically in cancer tissue.
- 請求項1から16のいずれかに記載の抗原結合分子及び医学的に許容し得る担体を含む、医薬組成物。 A pharmaceutical composition comprising the antigen-binding molecule according to any one of claims 1 to 16 and a medically acceptable carrier.
- 請求項1から16のいずれかに記載の抗原結合分子を製造する方法であって、工程(i)~(iv)を含む方法:
(i)第1の抗原又は第2の抗原に結合する抗体の可変領域の少なくとも1つのアミノ酸が改変された抗原結合分子であって、該改変された可変領域のアミノ酸の少なくとも1つが互いに異なる可変領域を含む抗原結合分子のライブラリーを作製する工程、
(ii)作製されたライブラリーの中から、第1の抗原及び第2の抗原に対して結合活性を有するが、該第1の抗原及び第2の抗原と同時には結合しない可変領域を含む抗原結合分子を選択する工程、
(iii)工程(ii)で選択された抗原結合分子の該可変領域をコードする核酸、及び/または、第3の抗原に結合する抗原結合分子の可変領域をコードする核酸を含む宿主細胞を培養して、第1の抗原と第2の抗原に結合することができるが該第1の抗原と第2の抗原とが同時には結合しない抗体の可変領域、及び/または、第3の抗原に結合する可変領域を含む、抗原結合分子を発現させる工程、並びに
(iv)前記宿主細胞培養物から抗原結合分子を回収する工程。 A method for producing an antigen-binding molecule according to any one of claims 1 to 16, comprising steps (i) to (iv):
(i) an antigen-binding molecule in which at least one amino acid of a variable region of an antibody that binds to the first antigen or the second antigen is modified, wherein at least one of the amino acids of the modified variable region is different from each other Creating a library of antigen binding molecules comprising the region,
(ii) an antigen comprising a variable region that has binding activity to the first antigen and the second antigen, but does not bind simultaneously with the first antigen and the second antigen, from the prepared library Selecting a binding molecule,
(iii) culturing a host cell containing a nucleic acid encoding the variable region of the antigen-binding molecule selected in step (ii) and / or a nucleic acid encoding the variable region of the antigen-binding molecule that binds to the third antigen Thus, the antibody can bind to the first antigen and the second antigen, but the first antigen and the second antigen do not bind simultaneously, and / or bind to the third antigen. Expressing an antigen-binding molecule comprising a variable region that:
(iv) recovering the antigen-binding molecule from the host cell culture. - 工程(ii)において選択する抗原結合分子に含まれる、第1の抗原と第2の抗原に同時には結合しない可変領域が、それぞれ異なる細胞上で発現している第1の抗原と第2の抗原に同時には結合しない可変領域である、請求項18に記載の製造方法。 The first and second antigens, which are included in the antigen-binding molecule selected in step (ii) and which are variable regions that do not bind to the first antigen and the second antigen at the same time, are expressed on different cells. The manufacturing method according to claim 18, which is a variable region that does not bind to each other simultaneously.
- 工程(iii)において培養する宿主細胞が、抗体のFc領域をコードする核酸をさらに含む、請求項18又は19に記載の製造方法。 The production method according to claim 18 or 19, wherein the host cell cultured in step (iii) further comprises a nucleic acid encoding the Fc region of the antibody.
- Fc領域のFcγRに対する結合活性が、天然型ヒトIgG1抗体のFc領域と比較して低下しているFc領域である、請求項20に記載の製造方法。 21. The production method according to claim 20, wherein the Fc region has a reduced Fc region binding activity to FcγR compared to the Fc region of a natural human IgG1 antibody.
- 製造する抗原結合分子が多重特異性抗体である、請求項18から21のいずれかに記載の製造方法。 The production method according to any one of claims 18 to 21, wherein the antigen-binding molecule to be produced is a multispecific antibody.
- 工程(i)における、可変領域の少なくとも1つの改変されたアミノ酸が、置換又は挿入されたアミノ酸である、請求項18から22のいずれかに記載の製造方法。 The production method according to any one of claims 18 to 22, wherein in step (i), at least one modified amino acid of the variable region is a substituted or inserted amino acid.
- 挿入されたアミノ酸の数が1~25個である、請求項23に記載の製造方法。 The production method according to claim 23, wherein the number of inserted amino acids is 1 to 25.
- 改変が、抗体の可変領域のCDR1、CDR2、CDR3又はFR3領域のアミノ酸の改変である、請求項18から24のいずれかに記載の製造方法。 25. The production method according to any one of claims 18 to 24, wherein the modification is modification of an amino acid of CDR1, CDR2, CDR3, or FR3 region of an antibody variable region.
- 改変が、ループ領域のアミノ酸の改変である、請求項18から25のいずれかに記載の製造方法。 The production method according to any one of claims 18 to 25, wherein the modification is modification of an amino acid in a loop region.
- 改変が、抗体のH鎖可変領域のKabatナンバリング31~35、50~65、71~74及び95~102、L鎖可変領域のKabatナンバリング24~34、50~56及び89~97から選ばれる少なくとも1つのアミノ酸の改変である、請求項18から25のいずれかに記載の製造方法。 The modification is at least selected from Kabat numbering 31-35, 50-65, 71-74 and 95-102 of the heavy chain variable region of the antibody, Kabat numbering 24-34, 50-56 and 89-97 of the light chain variable region The production method according to any one of claims 18 to 25, wherein the production method is modification of one amino acid.
- 第1の抗原又は第2の抗原のいずれか1つが、T細胞表面に特異的に発現している分子であり、他方の抗原が、T細胞又は他の免疫細胞表面に発現している分子である、請求項18から27のいずれかに記載の製造方法。 Either one of the first antigen and the second antigen is a molecule that is specifically expressed on the surface of T cells, and the other antigen is a molecule that is expressed on the surface of T cells or other immune cells. The manufacturing method according to any one of claims 18 to 27.
- 第1の抗原又は第2の抗原のいずれか1つがCD3であり、他方の抗原がFcγR、TLR、IgA、レクチン、免疫チェックポイント分子、TNFスーパーファミリー分子、TNFRスーパーファミリー分子又はNKレセプター分子である、請求項28に記載の製造方法。 Either one of the first antigen or the second antigen is CD3, and the other antigen is FcγR, TLR, IgA, lectin, immune checkpoint molecule, TNF superfamily molecule, TNFR superfamily molecule or NK receptor molecule The manufacturing method according to claim 28.
- 第3の抗原が、がん組織特異的に発現している分子である、請求項28又は29に記載の製造方法。 30. The production method according to claim 28 or 29, wherein the third antigen is a molecule expressed specifically in cancer tissue.
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JP2015546719A JPWO2015068847A1 (en) | 2013-11-11 | 2014-11-11 | Antigen-binding molecules comprising modified antibody variable regions |
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